Methods used to treat cancer

ABSTRACT

The invention encompasses methods of treating a patient with cancer, such as glioblastoma. The methods may include the administration of one or more pharmaceutical compositions that are capable of inhibiting TROY to treat the patient with cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 62/145,040, filed Apr. 9, 2015, and is acontinuation-in-part of U.S. patent application Ser. No. 14/595,423,filed Jan. 13, 2015, which claims the benefit of U.S. patent applicationSer. No. 13/846,056, filed Mar. 18, 2013, now abandoned, which claimsthe benefit of U.S. patent application Ser. No. 12/911,091, filed Oct.25, 2010, now abandoned, which claims the benefit of U.S. ProvisionalApplication No. 61/254,615, filed Oct. 23, 2009, now expired, thecontents each of which are incorporated herein and are not admitted tobe prior art with respect to the present invention by the mention inthis cross-reference section.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under N5086853 andCA108961 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Glioblastoma multiforme (GBM) is the most malignant form of all primaryadult brain tumors (See Reference 1) Although significant technicaladvances in surgical and radiation treatment for brain tumors haveemerged, their impact on clinical outcome for patients has been onlymodest (See References 2-4). Of the features that characterize GBM,arguably none is more clinically significant than the propensity ofglioma cells to infiltrate into normal brain tissue. These invasivecells render complete resection impossible and confer resistance tochemo- and radiation-therapy. Thus, improved treatment of malignantglioma awaits a way of targeting the dispersing tumor cells in the CNS.

BRIEF SUMMARY OF THE INVENTION

Some embodiments of the invention may include a method of treating apatient with cancer. For example, in some aspect, the method may includethe step of administering a therapeutically effective amount of a firstpharmaceutical composition to the patient with cancer. In someembodiments, the first pharmaceutical composition may includepropentofylline or a pharmaceutically acceptable salt and the cancer maybe glioblastoma (e.g., invasive glioblastoma). Moreover, the firstpharmaceutical composition may include one or more pharmaceuticallyacceptable carriers. In some embodiments, the methods of the currentinvention may also include the administration of a second pharmaceuticalcomposition, which may further comprise at least one of the following:TROY inhibitors, Pyk2 inhibitors, Rac1 inhibitors, Dock180 inhibitors,Dock7 inhibitors, temozolomide and bevacizumab. In some embodiments, themethod may also include the administration of therapeutically effectiveamounts of radiation to the patient. In addition, in some aspects, thesecond pharmaceutical composition may be co-administered to the patient.Moreover, in some embodiments, the second pharmaceutical composition maycomprise the administration of radiation to the patient with cancer.

Some embodiments of the invention provide a method of treatingglioblastoma cells (e.g., invasive glioblastoma cells) in a subject. Themethod may include the steps of contacting the glioblastoma cells with atherapeutically effective amount of propentofylline or apharmaceutically acceptable salt thereof and further contacting theglioblastoma cells with a pharmaceutical composition. In some aspects,the pharmaceutical composition may comprise the administration of one ormore compounds selected from the group consisting of Pyk2 inhibitors,Rac1 inhibitors, Dock180 inhibitors, Dock7 inhibitors, temozolomide andbevacizumab. In other aspects, the method of may include thepharmaceutical composition comprising a therapeutically effective amountof radiation. Moreover, in some embodiments, the method may includesequentially contacting the glioblastoma cells with the therapeuticallyeffective amount of propentofylline or a pharmaceutically acceptablesalt thereof and the pharmaceutical composition. Alternatively, in someembodiments, the method may include substantially simultaneouslycontacting the glioblastoma cells with the therapeutically effectiveamount of propentofylline or a pharmaceutically acceptable salt thereofand the pharmaceutical composition.

Some embodiments may further comprise a pharmaceutical composition forthe treatment of glioblastoma. The pharmaceutical composition mayinclude a therapeutically effective amount of first active ingredientcomprising propentofylline or a pharmaceutically acceptable salt thereofand a second active ingredient. By way of example only, the secondactive ingredient may comprise one or more compounds selected from thegroup consisting of Pyk2 inhibitors, Rac1 inhibitors, Dock180inhibitors, Dock7 inhibitors, temozolomide and bevacizumab. Moreover, insome embodiments, the pharmaceutical composition may further comprise atleast one pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

A more complete understanding of the present invention may be derived byreferring to the detailed description when considered in connection withthe following illustrative figures.

FIG. 1 depicts expression of TROY in normal brain and variousglioblastoma types in the NCBI Gene Expression Omnibus GDS1962 dataset.

FIG. 2 depicts expression of TROY in a separate set of normal brain andvarious glioblastoma types by QRT-PCR.

FIG. 3 depicts TROY expression data from the NCBI Gene ExpressionOmnibus GDS1962 dataset with tissues grouped as long-term and short-termsurvivors.

FIG. 4 depicts a Western blot showing expression of TROY in fourglioblastoma cell lines.

FIG. 5 depicts a Western blot showing suppression of TROY expression intwo of the cell types using siRNA targeting TROY.

FIG. 6 depicts a graph showing significantly slowed migration of fourglioblastoma cell lines when those lines are transfected with siRNAtargeting TROY.

FIG. 7 depicts expression of a construct comprising HA-tagged TROYtransfected into two glioblastoma cell lines.

FIG. 8 depicts increased migration rate of cell lines transfected withthe HA-tagged TROY construct.

FIG. 9 depicts increased depth of invasion into rat brain slices by aGFP-tagged TROY transfected cell line.

FIGS. 10A-10C depict immunofluorescent staining for HA in HA-tagged TROYtransfected cell lines. FIG. 10A depicts immunofluorescent staining forTROY in T98G cells using an anti-HA antibody. FIG. 10B depictsimmunofluorescent staining for TROY in T98G-TROY-HA cells using ananti-HA antibody. FIG. 10C represents T98G-TROY-HA stained withsecondary antibody alone. The arrows in FIG. 10B represent TROY stainingat the membrane periphery and cellular extension.

FIG. 11 depicts (top) a Western Blot showing cell lysates from aglioblastoma cell line transfected with Pyk2 or HA-tagged TROY asindicated and (bottom) a Western blot of immunoprecipitates with anti-HAantibodies that show an association of Pyk2 with TROY.

FIG. 12 depicts increased migration rate in a glioblastoma cell linetransfected with HA-tagged TROY that is slowed when Pyk2 expression issuppressed.

FIG. 13 depicts reduced migration rate in a glioblastoma cell linetransfected with a dominant-negative Pyk2 construct, whether or not theTROY expression is endogenous or from HA-tagged TROY.

FIG. 14 depicts a Western blot showing increased phosphorylation of RhoAwhen TROY expression is suppressed and reduced phosphorylation of Rac-1when TROY expression is suppressed.

FIG. 15 depicts a Western blot showing increased phosphorylation ofRac-1 when a cell line is transfected with HA-tagged TROY. This effectis reduced when Pyk2 expression is suppressed.

FIG. 16 depicts reduced migration of a glioblastoma cell line when Rac-1expression is suppressed—whether or not the cell line expressesendogenous or HA-tagged TROY.

FIG. 17 depicts a Western blot validation of suppression of Rac1expression by Rac1 siRNA using alpha tubulin as a loading control.

FIG. 18 depicts increased Akt, Ina, and Erk1/2 phosphorylation when aglioblastoma cell line is transfected with HA-tagged TROY.

FIG. 19 depicts increased sensitivity of a glioblastoma cell line totemozolomide when TROY expression is suppressed.

FIG. 20 depicts reduced sensitivity of a glioblastoma cell line totemozolomide when the cell line is transfected with an HA-tagged TROYconstruct.

FIG. 21 depicts reduced migration of a glioblastoma cell line whenDock180 and Dock7 expression are suppressed.

FIG. 22 depicts reduced depth of invasion of glioblastoma cells whenDock7 expression is suppressed using two different siRNAs.

FIG. 23 depicts a Western blot showing TROY expression in glioblastomaxenografts grown in murine brain.

FIGS. 24A-24D illustrate that PPF treatment decreases TROY expression inGBM cells. FIG. 24A—a Western blot analysis of TROY expression in T98Gcells. Cells were treated with PPF at indicated concentrations for 6hrs, lysed, and cell lysates immunoblotted with anti-TROY antibody.Immunoblotting of α-Tubulin in cell lysates is included as a loadingcontrol. FIG. 24B—Western blot analysis of TROY expression in T98G,GBM43, and GBM10 cells. Cells were treated with PPF at indicatedconcentrations for 24 hrs, lysed, and cell lysates immunoblotted withanti-TROY and α-Tubulin antibodies. FIG. 24C—Western Blot analysis ofTROY expression in GBM10 and GBM43 primary glioma cells. Cells weretreated with 5 μM PPF, lysed at the indicated time-points, and thelysates immunoblotted with an anti-TROY antibody. Immunoblotting ofβ-Actin in cell lysates is included as a loading control. FIG.24D—Western Blot analysis of T98G, GBM10 and GBM43 glioma cells treatedwith 5 μM PPF for 24 hours. Lysates were immunoblotted with anti-TROY,anti-TNFR1, anti-EGFR, and β-Actin antibodies.

FIGS. 25A and 25B illustrate that PPF does not affect the proliferationin GBM cells. FIG. 25A—T98G, GBM43, and GBM10 cells were incubated withincreasing concentrations of PPF (0, 5, 50, and 500 μM). After 0, 48, 96and 144 hours of treatment, cell were trypsinized and counted using anautomated cell counter. FIG. 24B—GBM43, GBM10, and T98G glioma cellswere treated with increasing doses of PPF (0.5, 1, 5, 10, and 20 μM) intriplicate. The Cell Titer Glo (Promega) reagent was used to measuresurvival. Raw values were normalized on a plate-by-plate basis such that100% cell viability was equivalent to the mean of vehicle wells and 0%cell viability was equivalent to the mean of the MG132 positive control.The normalized data was used to assess viability of glioma cells afterPPF treatment.

FIGS. 26A-26C depict that PPF sensitizes GBM cells to TMZ andradiotherapy. FIG. 26A—A clonogenic assay was used to assess T98G andGBM43 cells survival after TMZ and radiation treatment. Cells werepre-treated with 5 μM PPF for 24 hours, and then either treated with 250μM TMZ for 24 hours or exposed to 2 Gy radiation. Graph depicts thesurviving fraction in the treated cells compared to vehicle (VC) treatedor non-treated (NT) cells, **p<0.01. FIG. 26B—T98G glioma cells weretreated with vehicle, PPF (5 μM), TMZ (250 μM), and PPF in combinationwith TMZ. TMZ-induced apoptosis was assayed by immunoblot analysis ofcell lysates with an antibody to cleaved PARP. Immunoblotting forα-Tubulin was included as a loading control. FIG. 26C—T98G, GBM10, andGBM43 cells were treated with PPF (5 μM), lysed, and then immunoblottedto assess the activation of AKT and NF-κB. Immunoblotting for 3-Actin isincluded as a loading control.

FIGS. 27A and 27B depict that PPF suppresses GBM cell invasion and Rac1activation. FIG. 27A—T98G, GBM10, and GBM43 glioma cells were treatedwith 5 μM PPF and invasion was assayed over 24 hours utilizing aMatrigel invasion assay, *p<0.05. FIG. 27B—T98G glioma cells were serumstarved, pre-incubated with 5 μM PPF or vehicle for 1 hour, and thenstimulated with 10% FBS for 2-10 mins. Cell lysates were harvested andequal concentrations of protein were assessed for Rac1 activation.

FIGS. 28A and 28B depict that PPF suppresses GBM cell membrane ruffling.FIG. 28A—GBM43 cells were preincubated with 5 μM PPF or vehicle for 1hour prior to 10% FBS stimulation for 5 min. After FBS stimulation,cells were fixed, permeabilized, and stained for F-actin. For eachexperimental condition, at least 12 images were taken randomly.Arrowhead indicates membrane ruffles. FIG. 28B—Graph depicts the averagelamellipodia in T98G, GBM10, and GBM43 cells in the presence or absenceof 10% FBS with or without 5 μM PPF as indicated. Lamellipodia weretraced using Image J software. For each cell, the fraction of the cellperimeter that displayed lamellipodia was calculated, *p<0.05.

Elements and acts in the figures are illustrated for simplicity and havenot necessarily been rendered according to any particular sequence orembodiment.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the invention. It will beunderstood, however, by those skilled in the relevant arts, that thepresent invention may be practiced without these specific details. Inother instances, known structures and devices are shown or discussedmore generally in order to avoid obscuring the invention.

Gliomas, primary brain tumors that derive from glial support cells, arethe most common primary tumor of the adult central nervous system andwill result in an estimated 13,000 deaths in 2010 (See References 1, 3,11, and 12). Adult gliomas of astrocytic origin (astrocytomas) comprisea spectrum of neoplasms that are generally classified by WHO standardsinto low-grade benign tumors (i.e. juvenile pilocytic astrocytoma,diffuse astrocytoma) and high-grade malignant tumors (i.e. anaplasticastrocytoma and glioblastoma multiforme; GBM). Patients diagnosed withgrade IV GBM, the most aggressive malignant glioma, have a mediansurvival of 9-12 months after the onset of clinical symptoms (SeeReferences 11-13). Molecular analyses of glioma specimens haveidentified several common genetic alterations (e.g., p16INK4a deletion)and gene expression changes (e.g., EGFR overexpression) that maycontribute to glioblastoma formation (See References 14 and 15).

In general, gliomas are extremely difficult to treat using conventionalapproaches (See References 12-16.) This is primarily due to theintrinsic propensity of glioma cells to exit the tumor core and invadethe adjacent normal brain parenchyma (See References 3 and 4). Thesemigrating cells escape surgical resection and are poorly targeted byradiation or chemotherapy. They sometimes travel over long distances,frequently along blood vessel and fiber tracts, and then initiatesecondary tumor growth at their final destination. This distinguishinginvasive ability is not shared by nonglial cells that metastasize fromother primary tumor sites (e.g. breast) to brain tissue. The invasion ofglioma cells is likely triggered by a presently undefined signal orsignals that promote a cascade of cellular responses, including cellelongation, integrin-mediated cell attachment to extracellular matrix(ECM) molecules, the production and secretion of ECM-degrading enzymes,and cell movement (See References 17 and 18).

Migrating glioma cells exhibit decreased susceptibility to pro-apoptoticagents (See Reference 19) providing them with an additional mechanismfor resisting current radiological and chemotherapeutic treatmentmodalities.

TROY (TNFRSF19) is an orphan member of the TNFR superfamily that ishighly expressed in embryonic and adult CNS, and developing hairfollicles (See References 5-10). During mouse embryogenesis, TROY mRNAis detected in many developing tissues including the limb buds, eyelids,whiskers, mammary glands, epidermis, bronchial, tongue, dental andgastric epithelium as well as the germinal zones of the CNS includingthe ventricular zone and subventricular zone. However, in adult animals,TROY expression changes and is primarily restricted to hair folliclesand neuron-like cells in the cerebrum, cerebral cortex, developingolfactory system as well as dorsal root and retinal ganglion neurons(See References 5-10) In the peripheral nervous system, TROY functionsas a co-receptor for the ligand-binding Nogo-66 receptor 1 (NgR1) toform the TROY/NgR1/LINGO complex that activates the RhoA pathway toinhibit neurite outgrowth of dorsal root ganglion neurons in adult mice(See References 6 and 9). In humans, TROY mRNA is primarily expressed inthe brain and also the prostate, whereas low or undetectable levels areobserved in the heart, lung, liver, thymus, uterus, skeletal muscle,spleen, colon testis, kidney and peripheral blood lymphocytes (SeeReference 20). The reason or mechanism for this “switch-off” of TROYexpression after birth is unclear, but its strict control indicates thataberrant expression may be detrimental. Indeed, it has been recentlyreported that TROY is highly expressed in primary and metastaticmelanoma cells, but not in melanocytes found in normal skin biopsies andprimary skin cell cultures (See Reference 21).

Herein, the Inventor demonstrates that TROY serves as a target or markerof invasive glioblastoma, that its expression is linked to poortherapeutic outcome and that it serves as a marker of resistance totemozolomide and as a marker of sensitivity to classes of drugs thattreat glioblastoma by targeting pathways that contribute to glioma cellmigration and invasion.

A marker may be any molecular structure produced by a cell, expressedinside the cell, accessible on the cell surface, or secreted by thecell. A marker may be any protein, carbohydrate, fat, nucleic acid,catalytic site, or any combination of these such as an enzyme,glycoprotein, cell membrane, virus, cell, organ, organelle, or any uni-or multimolecular structure or any other such structure now known or yetto be disclosed whether alone or in combination. A marker may also becalled a target and the terms are used interchangeably.

A marker may be represented by the sequence of a nucleic acid from whichit can be derived. Examples of such nucleic acids include miRNA, tRNA,siRNA, mRNA, cDNA, or genomic DNA sequences. While a marker may berepresented by the sequence of a single nucleic acid strand (e.g.5′→3′), nucleic acid reagents that bind the marker may also bind to thecomplementary strand (e.g. 3′→5′). Alternatively, a marker may berepresented by a protein sequence. The concept of a marker is notlimited to the products of the exact nucleic acid sequence or proteinsequence by which it may be represented. Rather, a marker encompassesall molecules that may be detected by a method of assessing theexpression of the marker.

Examples of molecules encompassed by a marker include point mutations,silent mutations, deletions, frameshift mutations, translocations,alternative splicing derivatives, differentially methylated sequences,differentially modified protein sequences, truncations, soluble forms ofcell membrane associated markers, and any other variation that resultsin a product that may be identified as the marker. The followingnonlimiting examples are included for the purposes of clarifying thisconcept: If expression of a specific marker in a sample is assessed byRTPCR, and if the sample expresses an mRNA sequence different from thesequence used to identify the specific marker by one or morenucleotides, but the marker may still be detected using RTPCR, then thespecific marker encompasses the sequence present in the sample.Alternatively if expression of a specific marker in a sample is assessedby an antibody and the amino acid sequence of the marker in the samplediffers from a sequence used to identify marker by one or more aminoacids, but the antibody is still able to bind to the version of themarker in the sample, then the specific marker encompasses the sequencepresent in the sample.

Expression encompasses any and all processes through which materialderived from a nucleic acid template may be produced. Expression thusincludes processes such as RNA transcription, mRNA splicing, proteintranslation, protein folding, post-translational modification, membranetransport, associations with other molecules, addition of carbohydratemoeties to proteins, phosphorylation, protein complex formation and anyother process along a continuum that results in biological materialderived from genetic material whether in vitro, in vivo, or ex vivo.Expression also encompasses all processes through which the productionof material derived from a nucleic acid template may be actively orpassively suppressed. Such processes include all aspects oftranscriptional and translational regulation. Examples includeheterochromatic silencing, differential methylation, transcriptionfactor inhibition, any form of RNAi silencing, microRNA silencing,alternative splicing, protease digestion, posttranslationalmodification, and alternative protein folding.

Expression may be assessed by any number of methods used to detectmaterial derived from a nucleic acid template used currently in the artand yet to be developed. Examples of such methods include any nucleicacid detection method including the following nonlimiting examples,microarray analysis, RNA in situ hybridization, RNAse protection assay,Northern blot, reverse transcriptase PCR, quantitative PCR, quantitativereverse transcriptase PCR, quantitative real-time reverse transcriptasePCR, reverse transcriptase treatment followed by direct sequencing,direct sequencing of genomic DNA, or any other method of detecting aspecific nucleic acid now known or yet to be disclosed. Other examplesinclude any process of assessing protein expression including flowcytometry, immunohistochemistry, ELISA, Western blot, and immunoaffinitychromatograpy, HPLC, mass spectrometry, protein microarray analysis,PAGE analysis, isoelectric focusing, 2-D gel electrophoresis, or anyenzymatic assay. Methods of detecting expression may include methods ofpurifying nucleic acid, protein, or some other material depending on thetype of marker. Any method of nucleic acid purification may be used,depending on the type of marker. Examples include phenol alcoholextraction, ethanol extraction, guanidium isothionate extraction, gelpurification, size exclusion chromatography, cesium chloridepreparations, and silica resin preparation. Any method of proteinpurification may be used, also depending on the type of marker. Examplesinclude size exclusion chromatography, hydrophobic interactionchromatography, ion exchange chromatography, affinity chromatograpy(including affinity chromatography of tagged proteins), metal binding,immunoaffinity chromatography, and HPLC.

Nucleic acid amplification is a process by which copies of a nucleicacid may be made from a source nucleic acid. Nucleic acids that may besubjected to amplification may be from any source. In some nucleicamplification methods, the copies are generated exponentially. Examplesof nucleic acid amplification include but are not limited to: thepolymerase chain reaction (PCR), ligase chain reaction (LCR),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA),amplification with Qβ replicase, whole genome amplification with enzymessuch as φ29, whole genome PCR, in vitro transcription with any RNApolymerase, or any other method by which copies of a desired sequenceare generated.

Polymerase chain reaction (PCR) is a particular method of amplifyingDNA, generally involving the mixing of a nucleic sample, two or moreprimers, a DNA polymerase, which may be a thermostable DNA polymerasesuch as Taq or Pfu, and deoxyribose nucleoside triphosphates (dNTPs). Ingeneral, the reaction mixture is subjected to temperature cyclescomprising a denaturation stage, (typically 80-100° C.) an annealingstage with a temperature that is selected based on the meltingtemperature (Tm) of the primers and the degeneracy of the primers, andan extension stage (for example 40-75° C.) In real-time PCR analysis,additional reagents, methods, optical detection systems, and devices areused that allow a measurement of the magnitude of fluorescence inproportion to concentration of amplified DNA. In such analyses,incorporation of fluorescent dye into the amplified strands may bedetected or labeled probes that bind to a specific sequence during theannealing phase release their fluorescent tags during the extensionphase. Either of these will allow a quantification of the amount ofspecific DNA present in the initial sample. Often, the result of areal-time PCR will be expressed in the terms of cycle threshold (Ct)values. The Ct represents the number of PCR cycles for the fluorescentsignal from a real-time PCR reaction to cross a threshold value offluorescence. Ct is inversely proportional to the amount of targetnucleic acid originally present in the sample. RNA may be detected byPCR analysis by creating a DNA template from RNA through a reversetranscriptase enzyme.

Other methods used to assess expression include the use of natural orartificial ligands capable of specifically binding a marker. Suchligands include antibodies, antibody complexes, conjugates, naturalligands, small molecules, nanoparticles, or any other molecular entitycapable of specific binding to a marker. Antibodies may be monoclonal,polyclonal, or any antibody fragment including an Fab, F(ab)₂, Fv, scFv,phage display antibody, peptibody, multispecific ligand, or any otherreagent with specific binding to a marker. Ligands may be associatedwith a label such as a radioactive isotope or chelate thereof, dye(fluorescent or nonfluorescent), stain, enzyme, metal, or any othersubstance capable of aiding a machine or a human eye fromdifferentiating a cell expressing a marker from a cell not expressing amarker. Additionally, expression may be assessed by monomeric ormultimeric ligands associated with substances capable of killing thecell. Such substances include protein or small molecule toxins,cytokines, pro-apoptotic substances, pore forming substances,radioactive isotopes, or any other substance capable of killing a cell.

Differential expression encompasses any detectable difference betweenthe expression of a marker in one sample relative to the expression ofthe marker in another sample. Differential expression may be assessed bya detector, an instrument containing a detector, or by aided or unaidedhuman eye. Examples include but are not limited to differential stainingof cells in an IHC assay configured to detect a marker, differentialdetection of bound RNA on a microarray to which a sequence capable ofbinding to the marker is bound, differential results in measuring RTPCRmeasured in the number of PCR cycles necessary to reach a particularoptical density at a wavelength at which a double stranded DNA bindingdye (e.g. SYBR Green) incorporates, differential results in measuringlabel from a reporter probe used in a real-time RTPCR reaction,differential detection of fluorescence on cells using a flow cytometer,differential intensities of bands in a Northern blot, differentialintensities of bands in an RNAse protection assay, differential celldeath measured by apoptotic markers, differential cell death measured byshrinkage of a tumor, or any method that allows a detection of adifference in signal between one sample or set of samples and anothersample or set of samples.

The expression of the marker in a sample may be compared to a level ofexpression predetermined to predict the presence or absence of aparticular physiological characteristic. The level of expression may bederived from a single control or a set of controls. A control may be anysample with a previously determined level of expression. A control maycomprise material within the sample or material from sources other thanthe sample. Alternatively, the expression of a marker in a sample may becompared to a control that has a level of expression predetermined tosignal or not signal a cellular or physiological characteristic. Thislevel of expression may be derived from a single source of materialincluding the sample itself or from a set of sources. Comparison of theexpression of the marker in the sample to a particular level ofexpression results in a prediction that the sample exhibits or does notexhibit the cellular or physiological characteristic.

Prediction of a cellular or physiological characteristic includes theprediction of any cellular or physiological state that may be predictedby assessing the expression of a marker. Examples include the identityof a cell as a particular cell including a particular normal or cancercell type, the likelihood that one or more diseases is present orabsent, the likelihood that a present disease will progress, remainunchanged, or regress, the likelihood that a disease will respond or notrespond to a particular therapy, or any other outcome. Further examplesinclude the likelihood that a cell will move, senesce, apoptose,differentiate, metastasize, or change from any state to any other stateor maintain its current state.

Expression of a marker in a sample may be more or less than that of alevel predetermined to predict the presence or absence of a cellular orphysiological characteristic. The expression of the marker in the samplemay be more than 1,000,000×, 100,000×, 10,000×, 1000×, 100×, 10×, 5×,2×, 1×, 0.5×, 0.1× 0.01×, 0.001×, 0.0001×, 0.00001×, 0.000001×,0.0000001× or less than that of a level predetermined to predict thepresence or absence of a cellular or physiological characteristic.

The invention contemplates assessing the expression of the marker in anybiological sample from which the expression may be assessed. One skilledin the art would know to select a particular biological sample and howto collect said sample depending upon the marker that is being assessed.Examples of sources of samples include but are not limited to biopsy orother in vivo or ex vivo analysis of prostate, breast, skin, muscle,facia, brain, endometrium, lung, head and neck, pancreas, smallintestine, blood, liver, testes, ovaries, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney, placenta, or fetus.In some aspects of the invention, the sample comprises a fluid sample,such as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal fluid,stool, or urine. Samples include single cells, whole organs or anyfraction of a whole organ, in any condition including in vitro, ex vivo,in vivo, post-mortem, fresh, fixed, or frozen.

One type of cellular or physiological characteristic is the risk that aparticular disease outcome will occur. Assessing this risk includes theperforming of any type of test, assay, examination, result, readout, orinterpretation that correlates with an increased or decreasedprobability that an individual has had, currently has, or will develop aparticular disease, disorder, symptom, syndrome, or any conditionrelated to health or bodily state. Examples of disease outcomes include,but need not be limited to survival, death, progression of existingdisease, remission of existing disease, initiation of onset of a diseasein an otherwise disease-free subject, or the continued lack of diseasein a subject in which there has been a remission of disease. Assessingthe risk of a particular disease encompasses diagnosis in which the typeof disease afflicting a subject is determined. Assessing the risk of adisease outcome also encompasses the concept of prognosis. A prognosismay be any assessment of the risk of disease outcome in an individual inwhich a particular disease has been diagnosed. Assessing the riskfurther encompasses prediction of therapeutic response in which atreatment regimen is chosen based on the assessment. Assessing the riskalso encompasses a prediction of overall survival after diagnosis.

Determining the level of expression that signifies a physiological orcellular characteristic may be assessed by any of a number of methods.The skilled artisan will understand that numerous methods may be used toselect a level of expression for a particular marker or a plurality ofmarkers that signifies one or more particular physiological or cellularcharacteristics. In diagnosing the presence of a disease, a thresholdvalue may be obtained by performing the assay method on samples obtainedfrom a population of patients having a certain type of disease (cancerfor example), and from a second population of subjects that do not havethe disease. In assessing disease outcome or the effect of treatment, apopulation of patients, all of which have, a disease such as cancer, maybe followed for a period of time. After the period of time expires, thepopulation may be divided into two or more groups. For example, thepopulation may be divided into a first group of patients whose diseaseprogresses to a particular endpoint and a second group of patients whosedisease does not progress to the particular endpoint. Examples ofendpoints include disease recurrence, death, metastasis or other statesto which disease may progress. If expression of the marker in a sampleis more similar to the predetermined expression of the marker in onegroup relative to the other group, the sample may be assigned a risk ofhaving the same outcome as the patient group to which it is moresimilar.

In addition, one or more levels of expression of the marker may beselected that signify a particular physiological or cellularcharacteristic. For example, Receiver Operating Characteristic curves,or “ROC” curves, may be calculated by plotting the value of a variableversus its relative frequency in two populations. For any particularmarker, a distribution of marker expression levels for subjects with andwithout a disease may overlap. This indicates that the test does notabsolutely distinguish between the two populations with completeaccuracy. The area of overlap indicates where the test cannotdistinguish the two groups. A threshold is selected. Expression of themarker in the sample above the threshold indicates the sample is similarto one group and expression of the marker below the threshold indicatesthe sample is similar to the other group. The area under the ROC curveis a measure of the probability that the expression correctly indicatedthe similarity of the sample to the proper group. See, e.g., Hanley etal., Radiology 143: 29-36 (1982) hereby incorporated by reference.

Additionally, levels of expression may be established by assessing theexpression of a marker in a sample from one patient, assessing theexpression of additional samples from the same patient obtained later intime, and comparing the expression of the marker from the later sampleswith the initial sample or samples. This method may be used in the caseof markers that indicate, for example, progression or worsening ofdisease or lack of efficacy of a treatment regimen or remission of adisease or efficacy of a treatment regimen.

Other methods may be used to assess how accurately the expression of amarker signifies a particular physiological or cellular characteristic.Such methods include a positive likelihood ratio, negative likelihoodratio, odds ratio, and/or hazard ratio. In the case of a likelihoodratio, the likelihood that the expression of the marker would be foundin a sample with a particular cellular or physiological characteristicis compared with the likelihood that the expression of the marker wouldbe found in a sample lacking the particular cellular or physiologicalcharacteristic.

An odds ratio measures effect size and describes the amount ofassociation or non-independence between two groups. An odds ratio is theratio of the odds of a marker being expressed in one set of samplesversus the odds of the marker being expressed in the other set ofsamples. An odds ratio of 1 indicates that the event or condition isequally likely to occur in both groups. An odds ratio grater or lessthan 1 indicates that expression of the marker is more likely to occurin one group or the other depending on how the odds ratio calculationwas set up. A hazard ratio may be calculated by estimate of relativerisk. Relative risk is the chance that a particular event will takeplace. It is a ratio of the probability that an event such asdevelopment or progression of a disease will occur in samples thatexceed a threshold level of expression of a marker over the probabilitythat the event will occur in samples that do not exceed a thresholdlevel of expression of a marker. Alternatively, a hazard ratio may becalculated by the limit of the number of events per unit time divided bythe number at risk as the time interval decreases. In the case of ahazard ratio, a value of 1 indicates that the relative risk is equal inboth the first and second groups. A value greater or less than 1indicates that the risk is greater in one group or another, depending onthe inputs into the calculation.

Additionally, multiple threshold levels of expression may be determined.This can be the case in so-called “tertile,” “quartile,” or “quintile”analyses. In these methods, multiple groups can be considered togetheras a single population, and are divided into 3 or more bins having equalnumbers of individuals. The boundary between two of these “bins” may beconsidered threshold levels of expression indicating a particular levelof risk of a disease developing or signifying a physiological orcellular state. A risk may be assigned based on which “bin” a testsubject falls into.

A subject includes any human or non-human mammal, including for example:a primate, cow, horse, pig, sheep, goat, dog, cat, or rodent, capable ofdeveloping cancer including human patients that are suspected of havingcancer, that have been diagnosed with cancer, or that have a familyhistory of cancer. Methods of identifying subjects suspected of havingcancer include but are not limited to: physical examination, familymedical history, subject medical history including exposure toenvironmental factors, biopsy, or any of a number of imagingtechnologies such as ultrasonography, computed tomography, magneticresonance imaging, magnetic resonance spectroscopy, or positron emissiontomography.

Cancer cells include any cells derived from a tumor, neoplasm, cancer,precancer, cell line, malignancy, or any other source of cells that havethe potential to expand and grow to an unlimited degree. Cancer cellsmay be derived from naturally occurring sources or may be artificiallycreated. Cancer cells may also be capable of invasion into other tissuesand metastasis. Cancer cells further encompass any malignant cells thathave invaded other tissues and/or metastasized. One or more cancer cellsin the context of an organism may also be called a cancer, tumor,neoplasm, growth, malignancy, or any other term used in the art todescribe cells in a cancerous state.

Examples of cancers that could serve as sources of cancer cells includesolid tumors such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, and retinoblastoma.

Additional cancers that may serve as sources of cancer cells includeblood borne cancers such as acute lymphoblastic leukemia (“ALL,”), acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia (“AML”), acute promyelocytic leukemia(“APL”), acute monoblastic leukemia, acute erythroleukemic leukemia,acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia (“CML”), chronic lymphocytic leukemia (“CLL”), hairycell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenousleukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease,non-Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

The present invention further provides kits to be used in assessing theexpression of a particular RNA in a sample from a subject to assess therisk of developing disease. Kits include any combination of componentsthat facilitates the performance of an assay. A kit that facilitatesassessing the expression of an RNA may include suitable nucleicacid-based and immunological reagents as well as suitable buffers,control reagents, and printed protocols.

Kits that facilitate nucleic acid based methods may further include oneor more of the following: specific nucleic acids such asoligonucleotides, labeling reagents, enzymes including PCR amplificationreagents such as Taq or Pfu; reverse transcriptase, or one or more otherpolymerases, and/or reagents that facilitate hybridization. Specificnucleic acids may include nucleic acids, polynucleotides,oligonucleotides (DNA, or RNA), or any combination of molecules thatincludes one or more of the above, or any other molecular entity capableof specific binding to a nucleic acid marker. In one aspect of theinvention, the specific nucleic acid comprises one or moreoligonucleotides capable of hybridizing to the marker.

A specific nucleic acid may include a label. A label may be anysubstance capable of aiding a machine, detector, sensor, device, orenhanced or unenhanced human eye from differentiating a sample that thatdisplays positive expression from a sample that displays reducedexpression. Examples of labels include but are not limited to: aradioactive isotope or chelate thereof, a dye (fluorescent ornonfluorescent) stain, enzyme, or nonradioactive metal. Specificexamples include but are not limited to: fluorescein, biotin,digoxigenin, alkaline phosphatase, biotin, streptavidin, ³H, ¹⁴C, ³²P,³⁵S, or any other compound capable of emitting radiation, rhodamine,4-(4′-dimethylaminophenylazo) benzoic acid (“Dabcyl”);4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)(“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid(“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof or anyother compound that signals the presence of the labeled nucleic acid. Inone embodiment of the invention, the label includes one or more dyesoptimized for use in genotyping. Examples of such dyes include but arenot limited to: dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA,TAMRA, NED, dROX, PET, and LIZ.

An oligonucleotide is a reagent capable of binding a nucleic acidsequence. An oligonucleotide may be any polynucleotide of at least 2nucleotides. Oligonucleotides may be less than 10, less than 15, lessthan 20, less than 30, less than 40, less than 50, less than 75, lessthan 100, less than 200, less than 500, or more than 500 nucleotides inlength. While oligonucleotides are often linear, they may, depending ontheir sequence and conditions, assume a two- or three-dimensionalstructure. Oligonucleotides may be chemically synthesized by any of anumber of methods including sequential synthesis, solid phase synthesis,or any other synthesis method now known or yet to be disclosed.Alternatively, oligonucleotides may be produced by recombinant DNA basedmethods. One skilled in the art would understand the length ofoligonucleotide necessary to perform a particular task. Oligonucleotidesmay be directly labeled, used as primers in PCR or sequencing reactions,or bound directly to a solid substrate as in oligonucleotide arrays.

A nucleotide is an individual deoxyribonucleotide or ribonucleotidebase. Examples of nucleotides include but are not limited to: adenine,thymine, guanine, cytosine, and uracil, which may be abbreviated as A,T, G, C, or U in representations of oligonucleotide or polynucleotidesequence. Any molecule of two or more nucleotide bases, whether DNA orRNA, may be termed a nucleic acid.

An oligonucleotide used to detect to an allele may be affixed to a solidsubstrate. Alternatively, the sample may be affixed to a solid substrateand the nucleic acid reagent placed into a mixture. For example, thenucleic acid reagent may be bound to a substrate in the case of an arrayor the sample may be bound to a substrate as the case of a SouthernBlot, Northern blot or other method that affixes the sample to asubstrate. A nucleic acid reagent or sample may be covalently bound tothe substrate or it may be bound by some non covalent interactionincluding electrostatic, hydrophobic, hydrogen bonding, Van Der Waals,magnetic, or any other interaction by which an oligonucleotide may beattached to a substrate while maintaining its ability to recognize theallele to which it has specificity. A substrate may be any solid or semisolid material onto which a probe may be affixed, attached or printed,either singly or in the formation of a microarray. Examples of substratematerials include but are not limited to polyvinyl, polysterene,polypropylene, polyester or any other plastic, glass, silicon dioxide orother silanes, hydrogels, gold, platinum, microbeads, micelles and otherlipid formations, nitrocellulose, or nylon membranes. The substrate maytake any shape, including a spherical bead or flat surface.

A nucleotide is an individual deoxyribonucleotide or ribonucleotidebase. Examples of nucleotides include but are not limited to: adenine,thymine, guanine, cytosine, and uracil, which may be abbreviated as A,T, G, C, or U in representations of oligonucleotide or polynucleotidesequence.

In some aspects of the invention, the probe may be affixed to a solidsubstrate. In other aspects of the invention, the sample may be affixedto a solid substrate. A probe or sample may be covalently bound to thesubstrate or it may be bound by some non covalent interaction includingelectrostatic, hydrophobic, hydrogen bonding, Van Der Waals, magnetic,or any other interaction by which a probe such as an oligonucleotideprobe may be attached to a substrate while maintaining its ability torecognize the allele to which it has specificity. A substrate may be anysolid or semi solid material onto which a probe may be affixed, attachedor printed, either singly or in the formation of a microarray. Examplesof substrate materials include but are not limited to polyvinyl,polysterene, polypropylene, polyester or any other plastic, glass,silicon dioxide or other silanes, hydrogels, gold, platinum, microbeads,micelles and other lipid formations, nitrocellulose, or nylon membranes.The substrate may take any form, including a spherical bead or flatsurface. For example, the probe may be bound to a substrate in the caseof an array. The sample may be bound to a substrate as (for example) thecase of Southern Blots, Northern blots or other method that affixes thesample to a substrate.

Kits may also contain reagents that detect proteins, often through theuse of an antibody. These kits will contain one or more specificantibodies, buffers, and other reagents configured to detect binding ofthe antibody to the specific epitope. One or more of the antibodies maybe labeled with a fluorescent, enzymatic, magnetic, metallic, chemical,or other label that signifies and/or locates the presence ofspecifically bound antibody. The kit may also contain one or moresecondary antibodies that specifically recognize epitopes on otherantibodies. These secondary antibodies may also be labeled. The conceptof a secondary antibody also encompasses non-antibody ligands thatspecifically bind an epitope or label of another antibody. For example,streptavidin or avidin may bind to biotin conjugated to anotherantibody. Such a kit may also contain enzymatic substrates that changecolor or some other property in the presence of an enzyme that isconjugated to one or more antibodies included in the kit.

A kit may also contain an indication of a result of the use of the kitthat signifies a particular physiological or cellular characteristic. Anindication includes any guide to a result that would signal the presenceor absence of any physiological or cellular state that the kit isconfigured to predict. For example, the indication may be expressednumerically, expressed as a color or density of a color, expressed as anintensity of a band, derived from a standard curve, or expressed incomparison to a control. The indication may be communicated through theuse of a writing that may be contained physically in or on the kit (on apiece of paper for example), posted on the Internet, mailed to the userseparately from the kit, or embedded in a software package. The writingmay be in any medium that communicates how the result may be used topredict the cellular or physiological characteristic such as a printeddocument, a photograph, sound, color, or any combination thereof.

The invention further encompasses pharmaceutical compositions thatinclude one or more active pharmaceutical agents as ingredients. By wayof example only, in some aspects, the active pharmaceutical agent maycomprise a TROY inhibitor, such as propentofylline (PPF) orpharmaceutically salts thereof. In other embodiments, the activepharmaceutical agent may comprise an epidermal growth factor receptorinhibitor, such as, but not limited to gefitinib, erlotinib, cetuximab,lapatinib, panitumumab, vandetanib, afatinib, icotinib, zalutumumab,nimotuzumab, and matuzumab or any combination thereof, includingpharmaceutically acceptable salts.

According to some embodiments, PPF((3-methyl-1-(5-oxohexyl)-7-propyl-3,7-dihydro-1H-purine-2,6-dione) isan atypical synthetic methylxanthine compound that has been studiedextensively in preclinical models of CNS disorders and in Phase II andIII clinical trials for Alzheimer's disease and vascular dementia (SeeReferences 37-38). These clinical trials revealed the therapeuticefficacy, blood brain barrier permeability, and a minimal side effectprofile of PPF (See Reference 39). It has been demonstrated thatsystemic PPF treatment decreased tumor growth in a CNS-1 rat model viamodulation of microglia (See Reference 40). PPF has been shown toinhibit the migration of microglia by decreasing TROY expression, thussuppressing the activation of downstream effector molecules, such asPyk2 and Rac1 (See Reference 41). Targeting molecular drivers of glialcell invasion hampers the dispersion of GBM cells and enhances theirvulnerability to adjuvant, chemo, and radiation therapy (See Reference42). This application further incorporates by reference for all purposesthe following non-patent literature: H. Dhruv et al., Journal ofNeurooncology February 2016, Vol 126, pages 397-404.

Such pharmaceutical compositions may take any physical form necessarydepending on a number of factors including the desired method ofadministration and the physicochemical and stereochemical form taken bythe active pharmaceutical agent or pharmaceutically acceptable saltsthereof. Such physical forms include a solid, liquid, gas, sol, gel,aerosol, or any other physical form now known or yet to be disclosed.The concept of a pharmaceutical composition including an activepharmaceutical agent also encompasses the active pharmaceutical agentwithout any other additive. The physical form of the invention mayaffect the route of administration and one skilled in the art would knowto choose a route of administration that takes into consideration boththe physical form of the active pharmaceutical agent and the disorder tobe treated. Pharmaceutical compositions that include the activepharmaceutical agent may be prepared using methodology well known in thepharmaceutical art. A pharmaceutical composition that includes theactive pharmaceutical agent may include a second effective compound of adistinct chemical formula from the active pharmaceutical agent. Forexample, in some embodiments, the pharmaceutical composition can includea combination of any of the aforementioned or later disclosed activepharmaceutical agents described in this instant application. This secondeffective compound may have the same or a similar molecular target asthe target or it may act upstream or downstream of the molecular targetof the active pharmaceutical agent with regard to one or morebiochemical pathways.

Pharmaceutical compositions including the active pharmaceutical agentinclude materials capable of modifying the physical form of a dosageunit. In one nonlimiting example, the composition includes a materialthat forms a coating that holds in the active pharmaceutical agent.Materials that may be used in such a coating, include, for example,sugar, shellac, gelatin, or any other inert coating agent.

Pharmaceutical compositions including the active pharmaceutical agentmay be prepared as a gas or aerosol. Aerosols encompass a variety ofsystems including colloids and pressurized packages. Delivery of acomposition in this form may include propulsion of a pharmaceuticalcomposition including the active pharmaceutical agent through use ofliquefied gas or other compressed gas or by a suitable pump system.Aerosols may be delivered in single phase, bi-phasic, or tri-phasicsystems.

In some aspects of the invention, the pharmaceutical compositionincluding the active pharmaceutical agent is in the form of a solvate.Such solvates are produced by the dissolution of the activepharmaceutical agent in a pharmaceutically acceptable solvent.Pharmaceutically acceptable solvents include any mixtures of more thanone solvent. Such solvents may include pyridine, chloroform,propan-1-ol, ethyl oleate, ethyl lactate, ethylene oxide, water,ethanol, and any other solvent that delivers a sufficient quantity ofthe disclosed compound to treat the affliction without seriouscomplications arising from the use of the solvent in a majority ofpatients.

Pharmaceutical compositions that include the disclosed compound may alsoinclude a pharmaceutically acceptable carrier. Carriers include anysubstance that may be administered with the disclosed compound with theintended purpose of facilitating, assisting, or helping theadministration or other delivery of the active pharmaceutical agent.Carriers include any liquid, solid, semisolid, gel, aerosol or anythingelse that may be combined with the active pharmaceutical agent to aid inits administration. Examples include diluents, adjuvants, excipients,water, oils (including petroleum, animal, vegetable or synthetic oils.)Such carriers include particulates such as a tablet or powder, liquidssuch as oral syrup or injectable liquid, and inhalable aerosols. Furtherexamples include saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, and urea. Such carriers may further includebinders such as ethyl cellulose, carboxymethylcellulose,microcrystalline cellulose, or gelatin; excipients such as starch,lactose or dextrins; disintegrating agents such as alginic acid, sodiumalginate, Primogel, and corn starch; lubricants such as magnesiumstearate or Sterotex; glidants such as colloidal silicon dioxide;sweetening agents such as sucrose or saccharin, a flavoring agent suchas peppermint, methyl salicylate or orange flavoring, or coloringagents. Further examples of carriers include polyethylene glycol,cyclodextrin, oils, or any other similar liquid carrier that may beformulated into a capsule. Still further examples of carriers includesterile diluents such as water for injection, saline solution,physiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides, polyethylene glycols,glycerin, cyclodextrin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose, thickening agents, lubricating agents, andcoloring agents.

The pharmaceutical composition including the active pharmaceutical agentmay take any of a number of formulations depending on thephysicochemical form of the composition and the type of administration.Such forms include solutions, suspensions, emulsions, tablets, pills,pellets, capsules, capsules including liquids, powders,sustained-release formulations, directed release formulations,lyophylates, suppositories, emulsions, aerosols, sprays, granules,powders, syrups, elixirs, or any other formulation now known or yet tobe disclosed. Additional examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin,hereby incorporated by reference in its entirety.

Methods of administration include, but are not limited to, oraladministration and parenteral administration. Parenteral administrationincludes, but is not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,sublingual, intranasal, intracerebral, intraventricular, intrathecal,intravaginal, transdermal, rectal, by inhalation, or topically to theears, nose, eyes, or skin. Other methods of administration include butare not limited to infusion techniques including infusion or bolusinjection, by absorption through epithelial or mucocutaneous liningssuch as oral mucosa, rectal and intestinal mucosa. Compositions forparenteral administration may be enclosed in ampoule, a disposablesyringe or a multiple-dose vial made of glass, plastic or othermaterial.

Administration may be systemic or local. Local administration isadministration of the disclosed compound to the area in need oftreatment. Examples include local infusion during surgery; topicalapplication, by local injection; by a catheter; by a suppository; or byan implant. Administration may be by direct injection at the site (orformer site) of a cancer, tumor, or precancerous tissue or into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection. Intraventricular injection can be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration may be achieved byany of a number of methods known in the art. Examples include use of aninhaler or nebulizer, formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Thedisclosed compound may be delivered in the context of a vesicle such asa liposome or any other natural or synthetic vesicle.

A pharmaceutical composition formulated so as to be administered byinjection may be prepared by dissolving the active pharmaceutical agentwith water so as to form a solution. In addition, a surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants include any complex capable of non-covalentinteraction with the active pharmaceutical agent so as to facilitatedissolution or homogeneous suspension of the active pharmaceuticalagent.

Pharmaceutical compositions including the active pharmaceutical agentmay be prepared in a form that facilitates topical or transdermaladministration. Such preparations may be in the form of a liquidsolution, cream, paste, lotion, shake lotion, powder, emulsion,ointment, gel base, transdermal patch or iontophoresis device. Examplesof bases used in such compositions include petrolatum, lanolin,polyethylene glycols, beeswax, mineral oil, diluents such as water andalcohol, and emulsifiers and stabilizers, thickening agents, or anyother suitable base now known or yet to be disclosed.

Addition of a pharmaceutical composition to cancer cells includes allactions by which an effect of the pharmaceutical composition on thecancer cell is realized. The type of addition chosen will depend uponwhether the cancer cells are in vivo, ex vivo, or in vitro, the physicalor chemical properties of the pharmaceutical composition, and the effectthe composition is to have on the cancer cell. Nonlimiting examples ofaddition include addition of a solution including the pharmaceuticalcomposition to tissue culture media in which in vitro cancer cells aregrowing; any method by which a pharmaceutical composition may beadministered to an animal including intravenous, per os, parenteral, orany other of the methods of administration; or the activation orinhibition of cells that in turn have effects on the cancer cells suchas immune cells (e.g. macrophages and CD8+ T cells) or endothelial cellsthat may differentiate into blood vessel structures in the process ofangiogenesis or vasculogenesis.

Determination of an effective amount of the active pharmaceutical agentis within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein. The effective amountof a pharmaceutical composition used to affect a particular purpose aswell as a pharmacologically acceptable dose determined by toxicity,excretion, and overall tolerance may be determined in cell cultures orexperimental animals by pharmaceutical and toxicological procedureseither known now by those skilled in the art or by any similar methodyet to be disclosed. One example is the determination of the IC₅₀ (halfmaximal inhibitory concentration) of the pharmaceutical composition invitro in cell lines or target molecules. Another example is thedetermination of the LD₅₀ (lethal dose causing death in 50% of thetested animals) of the pharmaceutical composition in experimentalanimals. The exact techniques used in determining an effective amountwill depend on factors such as the type and physical/chemical propertiesof the pharmaceutical composition, the property being tested, andwhether the test is to be performed in vitro or in vivo. Thedetermination of an effective amount of a pharmaceutical compositionwill be well known to one of skill in the art who will use data obtainedfrom any tests in making that determination. Determination of aneffective amount of disclosed compound for addition to a cancer cellalso includes the determination of an effective therapeutic amount,including the formulation of an effective dose range for use in vivo,including in humans.

Treatment is contemplated in living entities including but not limitedto mammals (particularly humans) as well as other mammals of economic orsocial importance, including those of an endangered status. Furtherexamples include livestock or other animals generally bred for humanconsumption and domesticated companion animals.

The toxicity and therapeutic efficacy of a pharmaceutical compositionmay be determined by standard pharmaceutical procedures in cell culturesor animals. Examples include the determination of the IC₅₀ and the LD₅₀for a subject compound. The data obtained from these cell culture assaysand animal studies can be used in formulating a range of dosage for usein human. The dosage may vary depending upon the dosage form employedand the route of administration utilized.

The effective amount of the pharmaceutical composition to result in theslowing of expansion of the cancer cells would preferably result in aconcentration at or near the target tissue that is effective in slowingcellular expansion in cancer cells, but have minimal effects onnon-cancer cells, including non-cancer cells exposed to radiation orrecognized chemotherapeutic chemical agents. Concentrations that producethese effects can be determined using, for example, apoptosis markerssuch as the apoptotic index and/or caspase activities either in vitro orin vivo.

Treatment of a condition is the practice of any method, process, orprocedure with the intent of halting, inhibiting, slowing or reversingthe progression of a disease, disorder or condition, substantiallyameliorating clinical symptoms of a disease disorder or condition, orsubstantially preventing the appearance of clinical symptoms of adisease, disorder or condition, up to and including returning thediseased entity to its condition prior to the development of thedisease.

The addition of a therapeutically effective amount of the pharmaceuticalcomposition encompasses any method of dosing of a composition. Dosing ofthe disclosed compound may include single or multiple administrations ofany of a number of pharmaceutical compositions that include thedisclosed compound as an active ingredient. Examples include a singleadministration of a slow release composition, a course of treatmentinvolving several treatments on a regular or irregular basis, multipleadministrations for a period of time until a diminution of the diseasestate is achieved, preventative treatments applied prior to theinstigation of symptoms, or any other dosing regimen known in the art oryet to be disclosed that one skilled in the art would recognize as apotentially effective regimen. A final dosing regimen including theregularity of and mode of administration will be dependent on any of anumber of factors including but not limited to the subject beingtreated; the severity of the affliction; the manner of administration,the stage of disease development, the presence of one or more otherconditions such as pregnancy, infancy, or the presence of one or moreadditional diseases; or any other factor now known or yet to bedisclosed that affects the choice of the mode of administration, thedose to be administered and the time period over which the dose isadministered.

Pharmaceutical compositions that include the active pharmaceutical agentmay be administered prior to, concurrently with, or after administrationof a second pharmaceutical composition that may or may not include theactive pharmaceutical agent. If the compositions are administeredconcurrently, they are administered within one minute of each other. Ifnot administered concurrently, the second pharmaceutical composition maybe administered a period of one or more minutes, hours, days, weeks, ormonths before or after the pharmaceutical composition that includes thecompound Alternatively, a combination of pharmaceutical compositions maybe cyclically administered. Cycling therapy involves the administrationof one or more pharmaceutical compositions for a period of time,followed by the administration of one or more different pharmaceuticalcompositions for a period of time and repeating this sequentialadministration, in order to reduce the development of resistance to oneor more of the compositions, to avoid or reduce the side effects of oneor more of the compositions, and/or to improve the efficacy of thetreatment.

The invention further encompasses kits that facilitate theadministration of the pharmaceutical composition to a diseased entity.An example of such a kit includes one or more unit dosages of the activepharmaceutical agent. The unit dosage would be enclosed in a preferablysterile container and would be comprised of the disclosed compound and apharmaceutically acceptable carrier. In another aspect, the unit dosagewould comprise one or more lyophilates of the compound. In this aspectof the invention, the kit may include another preferably sterilecontainer enclosing a solution capable of dissolving the lyophilate.However, such a solution need not be included in the kit and may beobtained separately from the lyophilate. In another aspect, the kit mayinclude one or more devices used in administrating the unit dosages or apharmaceutical composition to be used in combination with the compound.Examples of such devices include, but are not limited to, a syringe, adrip bag, a patch or an enema. In some aspects of the invention, thedevice comprises the container that encloses the unit dosage.

Pharmaceutical compositions including the active pharmaceutical agentmay be used in methods of treating cancer. Such methods involve theadministration of a therapeutic amount of a pharmaceutical compositionthat includes the active pharmaceutical agent and/or a pharmaceuticallyacceptable salt thereof to a mammal, preferably a mammal in which acancer has been diagnosed.

A therapeutic amount further includes the prevention of progression ofthe cancer to a neoplastic, malignant or metastatic state. Suchpreventative use is indicated in conditions known or suspected ofpreceding progression to cancer, in particular, where non- orprecancerous cell growth consisting of hyperplasia, metaplasia, or mostparticularly, dysplasia has occurred (for review of such abnormal growthconditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B.Saunders Co., Philadelphia, pp. 68-90, incorporated by reference).Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or activity. For example, endometrialhyperplasia often precedes endometrial cancer and precancerous colonpolyps often transform into cancerous lesions. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. A typicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype or of a malignantphenotype, displayed in vivo or displayed in vitro by a cell samplederived from a patient can indicate the desirability ofprophylactic/therapeutic administration of the pharmaceuticalcomposition that includes the compound. Such characteristics of atransformed phenotype include morphology changes, looser substratumattachment, loss of contact inhibition, loss of anchorage dependence,protease release, increased sugar transport, decreased serumrequirement, expression of fetal antigens, disappearance of cell surfaceproteins, etc. Further examples include leukoplakia, featuring abenign-appearing hyperplastic or dysplastic lesion of the epithelium, orBowen's disease, a carcinoma in situ. Both of theses are pre-cancerouslesions indicative of the desirability of prophylactic intervention. Inanother example, fibrocystic disease including cystic hyperplasia,mammary dysplasia, adenosis, or benign epithelial hyperplasia isindicates desirability of prophylactic intervention.

In some aspects of the invention, use of the pharmaceutical compositionmay be determined by one or more physical factors such as tumor size andgrade or one or more molecular markers and/or expression signatures thatindicate prognosis and the likely response to treatment with thecompound. For example, determination of estrogen (ER) and progesterone(PR) steroid hormone receptor status has become a routine procedure inassessment of breast cancer patients. See, for example, Fitzgibbons etal, Arch. Pathol. Lab. Med. 124:966-78, 2000, incorporated by reference.Tumors that are hormone receptor positive are more likely to respond tohormone therapy and also typically grow less aggressively, therebyresulting in a better prognosis for patients with ER+/PR+ tumors. In afurther example, overexpression of human epidermal growth factorreceptor 2 (HER-2/neu), a transmembrane tyrosine kinase receptorprotein, has been correlated with poor breast cancer prognosis (see,e.g., Ross et al, The Oncologist 8:307-25, 2003), and Her-2 expressionlevels in breast tumors are used to predict response to the anti-Her-2monoclonal antibody therapeutic trastuzumab (Herceptin®, Genentech,South San Francisco, Calif.).

In another aspect of the invention, the diseased entity exhibits one ormore predisposing factors for malignancy that may be treated byadministration of a pharmaceutical composition including the compound.Such predisposing factors include but are not limited to chromosomaltranslocations associated with a malignancy such as the Philadelphiachromosome for chronic myelogenous leukemia and t (14; 18) forfollicular lymphoma; an incidence of polyposis or Gardner's syndromethat are indicative of colon cancer; benign monoclonal gammopathy whichis indicative of multiple myeloma, kinship with persons who have had orcurrently have a cancer or precancerous disease, exposure tocarcinogens, or any other predisposing factor that indicates inincreased incidence of cancer now known or yet to be disclosed.

The invention further encompasses methods of treating cancer thatcomprise combination therapies that comprise the administration of apharmaceutical composition including the disclosed compound and anothertreatment modality. Such treatment modalities include but are notlimited to, radiotherapy, chemotherapy (e.g., conventional chemotherapy,including the use of compounds such as temozolomide), surgery,immunotherapy, cancer vaccines, radioimmunotherapy, treatment withpharmaceutical compositions other than those which include the disclosedcompound, or any other method that effectively treats cancer incombination with the disclosed compound now known or yet to bedisclosed. Combination therapies may act synergistically. That is, thecombination of the two therapies is more effective than either therapyadministered alone. This results in a situation in which lower dosagesof both treatment modality may be used effectively. This in turn reducesthe toxicity and side effects, if any, associated with theadministration either modality without a reduction in efficacy.

In another aspect of the invention, the pharmaceutical compositionincluding the disclosed compound is administered in combination with atherapeutically effective amount of radiotherapy. The radiotherapy maybe administered concurrently with, prior to, or following theadministration of the pharmaceutical composition including the compound.The radiotherapy may act additively or synergistically with thepharmaceutical composition including the compound. This particularaspect of the invention would be most effective in cancers known to beresponsive to radiotherapy. Cancers known to be responsive toradiotherapy include, but are not limited to, Non-Hodgkin's lymphoma,Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer,ovarian cancer, bladder cancer, larynx cancer, cervical cancer,nasopharynx cancer, breast cancer, colon cancer, pancreatic cancer, headand neck cancer, esophageal cancer, rectal cancer, small-cell lungcancer, non-small cell lung cancer, brain tumors, other CNS neoplasms,or any other such tumor now known or yet to be disclosed.

Examples of pharmaceutical compositions that may be used in combinationwith the active pharmaceutical agent(s) may include commonchemotherapeutic agents/compositions such as cis-diamminedichloroplatinum (II) (cisplatin), doxorubicin, 5-fluorouracil, taxol, andtopoisomerase inhibitors such as etoposide, teniposide, irinotecan andtopotecan. For example, the chemotherapeutic agents might also includeTROY inhibitors, Pyk2 inhibitors, Rac1 inhibitors, Dock180 inhibitors,Dock7 inhibitors, temozolomide and bevacizumab. Still otherpharmaceutical compositions include antiemetic compositions such asmetoclopromide, domperidone, prochlorperazine, promethazine,chlorpromazine, trimethobenzamide, ondansetron, granisetron,hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron,benzquinamide, bietanautine, bromopride, buclizine, clebopride,cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine,methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine,thioproperazine and tropisetron.

Still other examples of pharmaceutical compositions that may be used incombination with the pharmaceutical composition including the disclosedcompound are hematopoietic colony stimulating factors. Examples ofhematopoietic colony stimulating factors include, but are not limitedto, filgrastim, sargramostim, molgramostim and epoietin alfa.Alternatively, the pharmaceutical composition including the disclosedcompound may be used in combination with an anxiolytic agent. Examplesof anxiolytic agents include, but are not limited to, buspirone, andbenzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate,clonazepam, chlordiazepoxide and alprazolam.

Pharmaceutical compositions that may be used in combination withpharmaceutical compositions that include the disclosed compound mayinclude analgesic agents. Such agents may be opioid or non-opioidanalgesic. Non-limiting examples of opioid analgesics inlcude morphine,heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon,apomorphine, normorphine, etorphine, buprenorphine, meperidine,lopermide, anileridine, ethoheptazine, piminidine, betaprodine,diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil,levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine,methadone, isomethadone and propoxyphene. Suitable non-opioid analgesicagents include, but are not limited to, aspirin, celecoxib, rofecoxib,diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen,ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid,nabumetone, naproxen, piroxicam, sulindac or any other analgesic nowknown or yet to be disclosed.

In other aspects of the invention, pharmaceutical compositions includingthe disclosed compound may be used in combination with a method thatinvolves treatment of cancer ex vivo. One example of such a treatment isan autologous stem cell transplant. In this method, a diseased entity'sautologous hematopoietic stem cells are harvested and purged of allcancer cells. A therapeutic amount of a pharmaceutical compositionincluding the disclosed compound may then be administered to the patientprior to restoring the entity's bone marrow by addition of either thepatient's own or donor stem cells.

Cancers that may be treated by pharmaceutical compositions including thedisclosed compound either alone or in combination with another treatmentmodality include solid tumors such as fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, and retinoblastoma.

Additional cancers that may be treated by pharmaceutical compositionsincluding the disclosed compound include blood borne cancers such asacute lymphoblastic leukemia (“ALL,”), acute lymphoblastic B-cellleukemia, acute lymphoblastic T-cell leukemia, acute myeloblasticleukemia (“AML”), acute promyelocytic leukemia (“APL”), acutemonoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia (“CML”), chronic lymphocytic leukemia (“CLL”), hairycell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenousleukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease,non-Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

Examples that represent different aspects of the invention follow. Suchexamples should not be construed as limiting the scope of thedisclosure. Alternative mechanistic pathways and analogous structureswithin the scope of the invention would be apparent to those skilled inthe art.

The invention encompasses inhibitors of cell migration activity andinhibitors of effector recruitment activity. Inhibition encompasses anyaction that hinders, from any detectable level up to and includingcomplete inactivation, the progression of a biological process. Suchbiological processes include expression of a gene or activities of agene product, progression of a disease, normal and abnormal metabolicactivities, interactions between entities within an organism, orinteractions between one organism and another. Further nonlimitingexamples of biological processes include development, death, maturation,infection, pain, apoptosis, or homeostasis. Inhibition includes actionsthat silence or repress the expression of a gene. Inhibition alsoincludes actions that hinder the activity of the RNA product, proteinproduct, or postranslationally modified protein product of a gene.Inhibition may be effectuated through a single agent that inactivates asingle gene or gene product, by a single agent that inactivates acombination of more than one gene or gene product, a combination ofagents that inactivates a single gene or gene product or a combinationof agents that inactivates a combination of more than one gene or geneproduct.

Inhibition may be effectuated directly by an agent that directly causesthe inhibition of a biological process or by agents that trigger one ormore different biological processes to effectuate the inhibition of thefirst biological process. Agents that cause inhibition may also becalled inhibitors. Examples of inhibitors include compositions such ascompounds that trigger RNAi silencing such as microRNA or siRNA, smallmolecular compounds, proteins such as soluble receptors or antibodies orany fragment thereof, including an Fab, F(ab)₂, Fv, scFv, Fc, phagedisplay antibody, peptibody or any other composition of matter that mayinactivate or hinder a biological process. Further nonlimiting examplesof inhibitors include X-rays, UV rays, visible light including laserlight, and sound.

Cell migration activity includes any mode through which a cell may movein two-dimensional or three-dimensional space. Such migration includesmovement through the use of pseudopodia including the adhesion ofpseudopodia to a surface, a flagellum, a cilium, acts of amoeboidmovement, extravasation, myosin-actin interactions, microtubuleextension, or any other process through which a cell moves itself fromone place to another or changes its morphology. In one aspect of theinvention, cell migration activity is measured through cell adhesion.Using adhesion, cell migration activity may be measured by cell-cellaggregation, monolayer radial migration, including adhesion to a cellmatrix comprising laminin, BSA or any other cell matrix component, threedimensional spheroid dispersion, or any other method that measuresadhesion based cellular migration in space. Migration activity may bemeasured by any method that detects that a cell has moved from one placeto another or has changed its morphology. Such methods include flowcytometry, capillary electrophoresis, visual examination by light,fluorescence, or electron microscopy, or any such method known in theart or yet to be developed. Inhibitors of cell migration activity areagents that disrupt any molecular or cellular process involved in cellmigration activity.

Effector recruitment activity includes any activity of a protein thatcontributes to the formation of a complex of two or more molecules thatserves to catalyze one or more chemical reactions. Effectors include anyprotein, nucleic acid or other molecule that may be included in acomplex that performs one or more biological activities. Recruitmentactivity encompasses any protein-protein interaction includingphosphorylation, dephosphorylation and other enzymatic activities,adhesion, signaling cascades, and cytokine/chemokine interactions, anyprotein-nucleic acid interactions, such as any of those involved intranscription, translation or DNA replication, or any other process thatincludes a protein interacting with another molecule. Inhibitors ofeffector recruitment activity may disrupt the interaction of a moleculewith any of the proteins listed above, the interaction between any ofthose proteins with each other, and further includes any members of acomplex that might be later identified.

In one aspect of the invention, inhibitors of effector recruitmentactivity may be identified on the basis of their ability to disrupt thebinding of a molecule to one or more of its effectors. This specificbinding may be measured by any method that allows the measurement of aprotein-protein interaction known in the art. Such method include thefollowing examples, alone or in combination as necessary:co-immunoprecipitation, biomolecular fluorescence complementation,fluorescence resonance energy transfer, label transfer, a yeasttwo-hybrid screen, in-vivo crosslinking, tandem affinity purification,chemical crosslinking, quantitative immunoprecipitation combined withknock-down (QUICK), dual polarization interferometry, protein-proteindocking, static light scattering, immunoprecipitation plusmass-spectrometry, Strep-protein interaction experiment (SPINE), surfaceplasmon resonance, fluorescence correlation spectroscopy, or any othermethod of measuring the specific interaction between one protein andanother now known in the art or yet to be disclosed.

In another aspect of the invention a glioblastoma patient is treated byfirst assessing the expression of a target and then treating with aneffective dose of an inhibitor of that target, potentially incombination with Temozolomide. The effective dose of a compound is thatamount effective to prevent occurrence of the symptoms of a disorder orto treat some symptoms of the disorder from which the patient suffers.Effective dose also includes an effective amount, a therapeutic amount,or any amount sufficient to elicit the desired pharmacological ortherapeutic effects, thus resulting in effective prevention or treatmentof the disorder. Thus, when treating a patient with glioblastoma, aneffective amount of compound is an amount sufficient to slow, or arrestthe progression, migration, metastasis, growth, or development of thetumor with the result that life is extended. Prevention includes a delayin onset of symptoms. Treatment includes a decrease in the symptomsassociated with the disorder or an amelioration of the recurrence of thesymptoms of the disorder. A pharmacologically acceptable doseencompasses any dose that may be administered to a patient that will notbe lethal to the patient or cause effects that threaten the health orthe life of the patient.

Patients include any human being, nonhuman primate, companion animal, ormammal suffering from a disease. In one aspect of the invention, thepatient has symptoms that signify the presence of a tumor or othergrowth in the brain. Such symptoms include headache, seizures, mental orpersonality changes, mass effect, or one of a number of focal orlocalized systems including ringing or buzzing sounds, hearing loss,loss of coordination, reduced sensation, weakness or paralysis,difficulty with walking or speech, difficulty keeping balance, decreasedmuscle control, or double vision. Patients may display one or moredifferent brain tumor types including acoustic neurinoma, astrocytoma,ependyoma, glioblastoma multiforme, meningioma, metastatic tumorsoriginating from another tumor type, mixed glioblastoma,oligodendroglioblastoma, or pineal region tumor.

Example

Elements and acts in the example are intended to illustrate theinvention for the sake of simplicity and have not necessarily beenrendered according to any particular sequence or embodiment. The exampleis also intended to establish possession of the invention by theInventors.

Members of the TNFR superfamily (TNFRSF), most notably TNFR1, have beenshown to play a role in inducing cell invasion and migration in severalcancer types. An expression microarray database containing 195clinically annotated brain tumor specimens publicly available at NCBI'sGene Expression Omnibus as dataset GSE4290 was analyzed. Snap-frozenspecimens from epileptogenic foci (NB, n=24), low-grade astrocytomas(LGA, n=29), and glioblastoma multiforme (GBM, n=82) with clinicalinformation were collected at the Hermelin Brain Tumor Center, HenryFord Hospital (Detroit, Mich.) as previously described (See Reference24). Gene expression profiling was conducted on all samples usingAffymetrix U133 Plus 2 GeneChips according to the manufacturer'sprotocol at the Neuro-Oncology Branch at the National Cancer Institute(Bethesda, Md.). For the analysis, gene expression data were normalizedin two ways: per chip normalization and per gene normalization acrossall samples in the collection. For per chip normalization, allexpression data on a chip were normalized to the 50th percentile of allvalues on that chip. For per gene normalization, the data for a givengene were normalized to the median expression level of that gene acrossall samples. Gene expression differences were deemed statisticallysignificant using parametric tests where variances were not assumedequal (Welch analysis of variance). Expression values were then filteredfor highly variable (differentially expressed) genes (coefficient ofvariation>30%) across samples producing a list of 7322 genes.TROY/TNFRSF19 expression is significantly differentially expressed amongbrain specimens. In normal brain specimens, TROY expression isrelatively low, but is increased with increasing tumor grade and issignificantly higher in GBM samples (n=82) (See FIG. 1). QuantitativeRT-PCR was performed on independent non-neoplastic (n=10), LGA (n=6),anaplastic astrocytoma (n=4), and GBM (n=22) specimens. Normal brainspecimens show relatively low mRNA levels for TROY as compared to thebrain tumor samples (p<0.01). In GBM specimens, the mRNA level of TROYis significantly higher than in normal brain (p<0.01) (See FIG. 2).Next, principal component analysis was done to discern possiblerelationships between subgroups of samples as in, for example, Reference34. Kaplan-Meier survival curves were developed for each principalcomponent cluster. One cluster had a median survival time of 401 days(short-term survival, ST) and the other cluster had a median survivaltime of 952 days (long-term survival, LT). Box-and-whisker plots forTROY expression level in each cluster derived from PC analysis weregraphed. Significance between the two populations was tested with atwo-sample t-test assuming unequal variances. Analysis of the Affymetrixexpression values for TROY in the GBM specimens for each cluster showedthat patients with GBM in the short-term survival cluster had higherexpression of TROY (10.5) than GBM patients in the long term survivalcluster (2.9; p<0.01) (See FIG. 3) This demonstrates that high TROYexpression levels correlates with poor patient outcome while low TROYexpression level corresponds with good patient outcome.

TROY is expressed in glioblastoma cell lines and siRNA-mediateddepletion of TROY suppresses glioblastoma cell migration. The expressionof TROY protein was assessed in four different cultured glioblastomacell lines. The highest level of expression of TROY was seen in U118cells, the next highest level of expression was seen in U87 cells, andthe lowest level of expression was seen in T98G and SNB19 cells (SeeFIG. 4).

RNAi was used to suppress the expression of TROY in each of the fourlisted glioma cell lines and the migratory behavior of the cells onglioma-derived ECM was examined using a two-dimensional radial cellmigration assay (See References 26 and 27). Suppression of TROY proteinexpression in all glioma cell lines was ˜80-90% effective with each oftwo independent siRNA oligonucleotides. Representative results are shownfor U118 cells (See FIG. 5). Further, suppression of TROY expression bysiRNA resulted in a significant (p<0.05) inhibition of cell migration inall four cell lines (See FIG. 18).

T98G and SNB19 glioma cell lines that stably express of HA-epitopetagged TROY were produced through lentiviral transduction. These wereused to further examine the role TROY signaling in glioma cell migration(See FIG. 7). Both the T98G and SNB19 lines normally express low levelsof endogenous TROY. The cell lines with HA tagged-TROY showed a˜1.8-2.3-fold increase in cell migration rate (See FIG. 8). Migration ofthe HA-tagged TROY expressing cells was further tested in the context ofan authentic brain microenvironment using an ex vivo organotypic ratbrain slice model. T98G glioma cells that overexpressed TROY displayed atwo-fold increase in the depth of cell invasion after 48 hours relativeto controls (See FIG. 9). Immunolocalization of TROY using an anti-HAantibody revealed that TROY was localized near the cell perimeter andwas enriched in lamellipodia (See FIG. 10B).

Potential effector molecules of TROY were found in immunoprecipitationexperiments coupled with MALDI-TOF MS analysis. In one experiment, T98Gcells expressing HA-tagged TROY and control T98G cells transfected withGFP were lysed, immunoprecipitated with anti-HA antibodies, and theimmunoprecipitates resolved by SDS-PAGE. Prominent protein bands presentin the immunoprecipitates of TROY expressing cells but absent in theimmunoprecipitates of control cells of interest were recovered from thegel. Proteins were eluted, and trypsin-digested. MALDI-TOF and MS-MSanalysis of the trypsin digests were performed on a Voyager reflectorinstrument (Applied Biosystems) and a Q-STAR mass spectrometer(Perceptive Biosystems) in positive ion mode.

The non-receptor protein tyrosine kinase Pyk2 was a candidate sequenceidentified by mass spectrometry in the TROY immunoprecipitate.Association of TROY with Pyk2 was verified by co-immunoprecipitation.T98G cells transfected with HA-tagged TROY or co-transfected withHA-tagged TROY and Pyk2 were immunoprecipitated with anti-HA antibodiesand the precipitates immunoblotted with anti-Pyk2 antibodies (See FIG.11). Both endogenous Pyk2 and transfected Pyk2 co-immunoprecipitatedwith TROY substantiating the intracellular interaction between TROY andPyk2.

Depletion of Pyk2 expression by shRNA in TROY overexpressing T98G cellswas performed to determine whether the association with Pyk2 wasrequired for TROY-induced stimulation of glioma migration. Suppressionof Pyk2 expression by shRNA significantly inhibited TROY stimulatedglioma cell migration (See FIG. 12). Further, coexpression of a kinaseinactive variant of Pyk2 (Pyk2KD) with TROY HA significantly inhibitedthe migration of the control T98G cells indicating that Pyk2 activity isrequired for TROY stimulated migration of glioma cells. (See FIG. 13).Finally, silencing of Pyk2 expression also inhibited TROY mediated Rac1activation (See FIG. 15). Together, these results indicate thatTROY-mediated glioma cell migration is dependent upon Pyk2 activity.

Rho GTPase family members, particularly Rac1 (See References 22-24, 28)effect the invasive behavior of glioblastoma cells. As a result, if TROYsignaling influences Rac1 activity, then TROY is a marker of invasiveglioblastoma and Rac1 is an effector molecule of TROY. U118 cellsexpress a high endogenous level of TROY protein expression (See FIG. 4)and display high Rac1 activity (See FIG. 14). Reduction of TROYexpression in U118 cells by siRNA resulted in decreased activity of Rac1(See FIG. 14). Further, siRNA mediated reduction of TROY expressioninduced RhoA activation, showing that TROY signaling modulates Rac1 andRhoA GTPases activity in opposite directions. Indeed, it has beenpreviously noted that in certain cell types, overexpression of TROYincreased RhoA activation (See References 6 and 9) suggesting that TROYsignaling may be modulated by cell type specific elements. To validatethe effect of TROY on Rac1 activity, the activation of Rac1 in gliomacells overexpressing TROY was compared to the activation of Rac1 inuntransfected cells. Overexpression of TROY resulted in a ˜2-foldinduction of Rac1 activation relative to untransfected cells (See FIG.15).

Since Pyk2 interacts with TROY and mediates TROY-induced migration, theeffect of Rac1 activation induced by TROY expression is dependent uponPyk2 activity was determined. shRNA-mediated depletion of Pyk2 in TROYoverexpressing glioma cells suppressed TROY induced Rac1 activity to thelevel of that in control cells. (See FIG. 15). This indicates that theTROY-mediated regulation of Rac1 activation is dependent upon Pyk2.Further, Rac1 expression in T98G cells overexpressing the TROY receptorwas reduced by Rac1 siRNA. That reduction in Rac1 expression in was ˜90%effective in T98G cells and caused a significant inhibition ofTROY-mediated cell migration. (See FIG. 16).

A recent study suggest that TROY is activated by the TNF family ligandlymphotoxin-α to induce NFκB activation, whereas previous studies havenot revealed specific interactions between TROY and any of the TNFfamily members (See Reference 29). Since Rac1 can influence multipledownstream signaling pathways, immunoblot analysis of lysates from TROYoverexpressing cells were analyzed for detection of various signalingpathways and compared to lysates from untransfected cells. Increasedphosphorylation of Akt, IκBα, and ERK1/2 in TROY overexpressing cellswas observed relative to untransfected cells (See FIG. 18).

Activation of Akt and NFκB signaling pathways plays a critical role incell survival. The effect of TROY expression on chemotherapy-inducedapoptosis in glioma cells was then determined by comparing thesensitivity of control U118 glioma cells and U118 glioma cells reducedTROY expression by transfection of TROY specific RNAi to temozolomidetreatment. U118 cells with reduced expression of TROY were significantlymore sensitive to cell death following temozolomide treatment relativeto U118 cells transfected with a negative RNAi control (See FIG. 19).Conversely, T98G glioma cells overexpressing TROY were significantlymore resistant to temozolomide induced apoptosis relative to controltransfected T98G glioma cells (See FIG. 20). Together, these dataindicate that TROY stimulated glioma cell migration/invasion increasesresistance to chemotherapy-induced cell death in glioma.

A number of RhoGTPases, including Rac1 Rac3 and Cdc42 (See References22-25), contribute to glioblastoma cell invasion in vitro. The RhoGTPases are activated by GEFs. There are currently 80 RhoGEFs in thehuman genome. Of these GEFs, 26 are known Rac1 activators, andcurrently, it is not known which Rac GEFs contribute to Rac1 activity inglial tumors. Rac GEFs that mediate glioma invasion were identified byfirst mining the NCBI expression microarray database of human braintumor specimens for the 26 GEFs known to have Rac exchange factoractivity. Of the 26 GEFs, Ect2, Trio and Vav3 exhibited increasedexpression in glioblastomas (GBMs) versus normal brain. Depletion ofEct2, Trio, and Vav3 expression reduced Rac1 activity in glioblastomacells which in turn led to a subsequent inhibition of glioblastoma cellmigration and invasion. A library of small interfering RNAs (siRNAs)directed against all 26 Rac GEFs in the human genome was used toevaluate the role of RacGEF's in inhibiting glioma invasion in a 96-wellformat invasion assay. Two additional Rac GEFs—Dock180 and Dock7—werefound to contribute to glioma invasion. Knockdown of Dock180 or Dock7expression by RNAi significantly reduced glioma invasion in vitro (SeeFIG. 21). Dock180 has recently been reported (See Reference 30) to beoverexpressed in invasive glioma cells where it regulates Rac1 activityand glioma cell invasion. To further examine the role of Dock7 inglioblastoma cell invasion using RNAi sequences that inhibit Dock 7expression to inhibit the migration of SNB19 cells into rat brainslices, a well-established ex vivo organotypic model for gliomainvasion. Knockdown of Dock7 expression significantly inhibited invasionrelative to control cells (See FIG. 22).

A significant limitation of the use of long-term established humanglioma cell lines for orthotopic xenografts is their propensity to formdiscrete, non-invasive tumors with well circumscribed borders that pushinto the adjacent normal brain tissue (See References 31-33). This is incontrast to the diffuse highly infiltrative growth that defines primaryGBM in patients. More important is the loss of genetic features andsignatures in long-term established cell lines which are common toprimary GBM. A model based on utilizing primary glioma xenograftsestablished and maintained by direct heterotypic transplantation,propagation, and passaging of patient tumor surgical samples in immunedeficient mice has been established. Intracranial tumors establishedwith these GBM xenografts retain key histopathological characteristicsof the aggressive behavior of the patients' tumors including localinvasion at the tumor periphery and invasion along white matter tracks,as well as manifesting key genetic features such as preservation of EGFRamplification status. Therefore, tumors that arise from these xenograftlines adequately model primary GBM in patients (See References 34-36).TROY protein expression was examined in lysates obtained from 19xenografts grown orthotopically in murine brain. Examination of TROYexpression on the xenograft lysates showed a range of TROY expression.Representative immunoblots showing GBM xenografts with high TROYexpression (GBM10), intermediate levels of TROY expression (GBM6, GBM8),or low levels of TROY expression (GBM44, GBM46) are shown in FIG. 23.

Referring now to FIG. 1: TROY mRNA expression levels derived from theNCBI Gene Expression Omnibus GDS1962 dataset are presented asbox-and-whisker plots. The box for each gene indicates the interquartilerange (25-75th percentile) and the line within this box indicates themedian value. Bottom and top bars of the whisker indicate the 10th and90th percentiles, respectively. Outliers are represented by closedcircles. Significance between the indicated classes of brain specimenswas tested using a two-sample t test assuming unequal variances.(NB=non-neoplastic brain; OL, Oligodendrogliomas; Astro=low gradeastrocytomas; GBM=glioblastoma multiforme). Referring now to FIG. 2Quantitative real-time PCR analysis of TROY expression in non-neoplasticbrain (NB), grade 1 low grade astrocytoma (LGA), grade 2-3 Astrocytomas(Astro) and glioblastoma multiforme (GBM) indicates that a higher levelof TROY expression signifies increased tumor grade. Values werenormalized to histone H3.3 and HPRT1 reference genes. Data are presentedas box-and-whisker plots. Referring now to FIG. 3: principal componentanalysis of brain tumors from NCBI Gene Expression Omnibus GDS1962dataset revealed two groups differing by their survival and were denotedas long term (LT) survival and short-term (ST) survival. These indicatethat a higher level of TROY expression signifies an association withshort-term survival. Box-and-whisker plots for TROY expression in GBMspecimens for each cluster are shown. Significance between the twopopulations was tested with a two-sample t test assuming unequalvariances.

Referring now to FIG. 4: T98G, SNB19, U87, and U118 cell lysates wereanalyzed for endogenous level of TROY expression by immunoblotting. Allexpress TROY with U87 and U118 cells having the highest expressionlevel. The levels of α-tubulin protein were also immunoblotted to ensureequal sample loading. Referring now to FIG. 5: knockdown of TROYexpression in U118 cells by two independent siRNA oligonucleotides. Notereduced expression of TROY protein in TROY-1 and TROY-2 transfected U118cells. Referring now to FIG. 18: the migration rate of each of the fourcell lines was slowed when the cell lines were transfected with siRNAoligonucleotides targeting TROY. siRNA targeting luciferase was used asa negative control. Migration rate was determined after 24 h migrationon glioma derived ECM (*−p<0.05).

Referring now to FIG. 7: Lysates of T98G or SNB19 cells transduced withempty lentiviral vector (v) or lentiviral vector encoding HA-epitopetagged TROY immunoblotted with anti-HA antibody shows that TROY isoverexpressed in cell lines transfected with the TROY construct.Referring now to FIG. 8: the migration rate of HA-TROY expressing gliomacells is faster than that of cells transduced with a negative controlconstruct. Cell migration was assessed over 48 h. Data represents theaverage of three independent experiments (*, p<0.01; **, p<0.05).Referring now to FIG. 9: T98G cells stably expressing green fluorescentprotein were transduced with lentiviruses expressing HA-tagged TROY.Cells were implanted into the bilateral putamen on rat organotypic brainslices and observed at 48 h. Depth of invasion was calculated fromZ-axis images collected by confocal laser scanning microscopy. The meanvalue of the depth of invasion was obtained from six independentexperiments (*, p<0.01). Cell lines transduced with a constructcontaining TROY displayed significantly greater depth of invasion.Referring now to FIGS. 10A, 10B, and 10C: immunofluorescent staining forTroy in T98G-Troy-HA cells using an anti-HA antibody shows that TROYlocalizes at the membrane periphery and within cellular extensions.

Referring now to FIG. 11: The top panel indicates that lysates ofnegative control transfected T98G cells, T98G cells transfected withHA-TROY, T98G cells transfected with Pyk2, or T98G cells cotransfectedwith HA-TROY and Pyk2 show expression of the transfected constructs whenimmunoblotted with anti-Pyk2 or anti-HA antibodies. In the bottom panel,the same cell lines were immunoprecipitated with anti-HA antibody andthe precipitates immunoblotted with anti-HA or anti-Pyk2 indicating thatPyk2 associates with TROY. Referring now to FIG. 12: The inhibition ofPyk2 expression by RNAi targeting Pyk2 suppresses Troy-induced gliomamigration. Migration rate of T98G, T98G-Troy-HA, and T98G-Troy-HA cellstransfected with a shRNA targeting Pyk2 was assessed over 24 h using aradial migration assay on 10 μg/ml laminin substrate (*, p<0.01). T98Gcells overexpressing TROY migrate at a faster rate than T98G cells thatlack the TROY expressing construct. This effect is negatived bytransfection with Pyk2-specific shRNA. Referring now to FIG. 13:inhibition of Pyk2 activity inhibits Troy-induced glioma migration. T98Gor T98G-Troy-HA expressing cells were infected with recombinantadenoviruses expressing a Pyk2 variant lacking the Pyk2 kinase domain.(Pyk2KD). Cell migration was assessed over 24 h using a radial migrationassay on 10 μg/ml laminin substrate (*, p<0.01). Transfection of thePyk2KD construct into T98G cells that do not overexpress TROY slowed themigration rate of those cells. T98G cells overexpresing TROY migrate atan even faster rate, but this effect is mitigated by transfection withPyk2KD.

Referring now to FIG. 14: U118 cells were left untransfected (NT),transfected with an siRNA targeting nonmammalian luciferase (ctrl), oran siRNA targeting TROY (Troy-1). Cells were cultured under serum-freemedium for an additional 16 hr prior to RhoA and Rac1 activation assays.Immunoblots show that RhoA is more likely to be phosphorylated and Rac-1is dephosphorylated upon suppression of TROY1 expression. Referring nowto FIG. 15: T98G and T98G-Troy-HA cells were transfected with a shRNAtargeting Pyk2 and cultured under serum-free medium for 16 h. Lysateswere then analyzed for activation of Rac1. Overexpression of TROY leadsto more Rac1 phosphorylation. This effect is diminished when Pyk2expression is suppressed. Referring now to FIG. 16: suppression of Rac1expression by siRNA suppresses Troy-induced glioma cell migration. T98Gand T98G-TROY-HA cells were transfected with an siRNA oligonucleotidetargeting Rac1. Cell migration was assessed over 24 h using a radialmigration assay on 10 mg/ml laminin substrate (*, p<0.01). Suppressionof Rac1 expression reduced the migration rate of the cells indicatingthat TROY-1 mediated migration works through Rac1. Referring now to FIG.17: a Western blot validating suppression of Rac1 and HA-TROY expressionwas performed.

Referring now to FIG. 18: TROY overexpression induces activation of Akt,NFkB and Erk1/2 signaling pathways. Cellular lysates of T98G gliomacells or T98G cells overexpressing TROY (left panel) and SNB19 gliomacells or SNB19 cells overexpressing TROY (right panel) wereimmunoblotted with the indicated antibodies. Equal sample loading wasverified by immunoblotting lysates with an anti-α-tubulin antibody.Western blots indicate phosphorylation of Akt, IκBα, and Erk1/2.

Referring now to FIG. 19: U118 cells were transfected with a siRNAtargeting TROY (Troy-1) or a siRNA targeting a nonmammalian gene. Cellswere then treated with 250 μM of temozolomide (TMZ) or vehicle (DMSO)for 48 h. The percentage of cell viability was measured by Alamar Blueassay and normalized to the control siRNA untreated with TMZ (*,p<0.01;**, p<0.001). The results indicate that when TROY expression issuppressed, the cells are rendered sensitive to temozolomide. Referringnow to FIG. 20: T98G and T98G expressing TROY-HA were treated with 250μM of TMZ or vehicle (DMSO) for 48 h. The percentage of cellularapoptosis was measured by annexin V staining followed by flow cytometry.Data represents the mean and S.D. from three independent experimentswith each experiment conducted in triplicate (**, p<0.001). The resultsshow that overexpression of TROY decreases the number of cells thatapoptose upon treatment with temozolomide and that overexpression ofTROY increases resistance to temozolomide.

Five known Rac1 activators were assessed for their ability to contributeto glioma invasion identified in a focused RhoGEF genome-wide siRNAscreening approach. Referring now to FIG. 21: SNB19 cells transfectedwith siRNAs targeting luciferase (ctrl), Dock180, or Dock7 were platedin transwell invasion chambers coated with Matrigel, and 24 h later,cells that had migrated through the filter were stained and counted.Shown are the mean of at least two independent experiments; bars, ±SE(*, p<0.001). Results indicate that Dock180 and Dock7 are implicated inthe invasive phenotype of glioma cells. Referring now to FIG. 22:SNB19-GFP cells were transfected with a siRNA targeting luciferase(ctrl) or two independent siRNAs targeting Dock7. After 24 h, cells wereimplanted into the bilateral putamen on rat organotypic brain slices andcultured for 48 h. Depth of invasion was calculated from Z-axis imagescollected by confocal laser scanning microscopy. The mean value of thedepth of invasion (+/−SEM) was obtained from three independentexperiments, performed in triplicates (*, p<0.01). The results furtherindicate a role of Dock7 in the development of an invasive phenotype.

Referring now to FIG. 23: Total cellular lystates from glioblastomamultiforme xenografts grown in murine brain orthopically were collectedand immunoblotted for human TROY. Ponceau staining was used as a loadingcontrol.

The following methodologies were used according to some embodiments ofthe invention and in conjunction with the experiments further detailedherein.

Cell Culture Conditions: Human glioma cell line T98G (ATCC) wasmaintained in DMEM with high glucose (Invitrogen) supplemented with 10%heat-inactivated fetal bovine serum (FBS) (Invitrogen) in a 37° C., 5%CO₂ atmosphere at constant humidity. The primary glioma patient derivedxenograft (PDX) lines GBM10 and GBM43 were derived from patient surgicalsamples and maintained as flank xenografts in immunodeficient mice.GBM10 and GBM43 flank tumor were resected, brought to single cellsuspension via mechanical dissociation, and maintained in DMEM+10% FBSfor in vitro experiments. In the experiments with FBS stimulation, thecells were serum starved (DMEM+0.1% Bovine Serum Albumin (BSA)) for 16hours before stimulation with 10% FBS in DMEM.

Antibodies and Reagents: PPF, TMZ, and laminin were purchased fromSigma-Aldrich. Antibody against TROY was obtained from Abcam®.Antibodies against EGFR, TNFR1, Fn14, phospho-NF-κB, NF-κB, phospho-AKT,AKT, Cleaved PARP, α-Tubulin, and β-Actin were purchased from CellSignaling Technology.

Referring now to FIGS. 24A, 24B, 24C, and 24D: The inventorsinvestigated if PPF could be utilized pharmacologically to decrease TROYexpression in GBM cells. The long-term established cell line T98G wasfirst utilized to test the efficacy of PPF. Cells were treated withincreasing concentration of PPF, 1 to 20 μM, lysed, and TROY expressionassessed by immunobloting. Briefly, monolayers of cells were washed inphosphate-buffered saline (PBS) containing 1 mMphenylmethylsulfonylfluoride and 1 mM sodium orthovanadate and thenlysed in 2×SDS sample buffer containing protease and phosphataseinhibitors. Protein concentrations were determined using the BCA Assay(Pierce). Thirty micrograms of total protein was loaded per lane andseparated by SDS-PAGE. After transfer, the nitrocellulose membrane(Invitrogen) was blocked with either 5% nonfat-milk or 5% BSA in TBSTbefore addition of primary antibodies and followed withperoxidase-conjugated secondary antibody (Promega). Protein bands weredetected using SuperSignal Chemiluminescent Substrate (Pierce) with aUVP BioSpectrum 500 Imaging System. TROY expression decreased inconcentration dependent manner, with significant suppression observedeven at 1 μM PPF concentration (FIG. 24A). The inventors furthervalidated these findings in patient-derived primary cell lines GBM10 andGBM43, which overexpress TROY, and showed that 5 μM PPF concentrationwas effective in lowering TROY expression in all glioma cells lines(FIG. 24B). TROY expression also decreased in a time-dependent manner,when treated with 5 μM PPF for varying lengths of time, in both GBM10and GBM43 cells (FIG. 24C). PPF specifically decreased TROY expressionin these glioma cells lines and did not affect the expression of othercell surface receptors, including the related TNFR1 or EGFR (FIG. 24D).This data demonstrates that PPF decreases TROY expression in GBM cellsin vitro.

Referring now to FIGS. 25A and 25B: To determine if PPF influencesglioma cell proliferation, the inventors treated T98G, GBM10, and GBM43cells with increasing concentrations of PPF and evaluated cellproliferation over the course of 144 hours. Briefly, 1.25×10⁵ cells wereseeded (n=3) in 12-well plates in 1 mL of DMEM supplemented with 10% FBSand allowed to attach at 37° for 16 hrs. Subsequently, the cells weretreated with media alone, 5, 50, and 500 μM PPF. After 0, 48, 96 and 144hours of treatment, the cells were trypsinized and counted using theautomated cell counter. PPF did not affect the proliferation of gliomacells at doses up to 500 μM (FIG. 25A). The inventors also investigatedwhether PPF induces glioma cell cytotoxicity using CellTiterGlo® assaywith increasing concentrations of PPF for 72 hours. Briefly, cells wereseeded at a density of 3000 cells/well (100 μL) in 96 well plates.Increasing concentrations of PPF (0.5 to 20 μM) were added to thedifferent wells (n=8) and incubated for 72 hours at 37° C. Subsequently,100 μL of CellTiterGlo® reagent was added to each well and luminescencewas measured using Envision Reader. On all 96 well plates, wellscontaining vehicle only or the positive control compound MG132 (aproteasome inhibitor) were also included. Raw values were normalized ona plate-by-plate basis such that 100% cell viability was equivalent tothe mean of vehicle wells and 0% cell viability was equivalent to themean of the MG132 positive control. The normalized data was used toassess viability of glioma cells after PPF treatment. Treatment with PPFresulted in a negligible loss of cell viability in all three GBM celllines (FIG. 25B). Together, these data corroborate the published reportsdescribing the limited side effect profile of PPF and demonstrate that,even at high doses, treatment with PPF does not cause toxicity in GBMcells (See Reference 39).

Referring now to FIGS. 26A, 26B, and 26C: Knockdown of TROY expressionwith shRNA decreases TMZ resistance in GBM cells in vitro (See Reference42). To corroborate these results with pharmacological inhibition ofTROY expression, the inventors tested whether the combination of PPFtreatment and the current standard of care would result in enhancedtherapeutic efficacy. Briefly, 5.0×10⁵ cells were seeded in 100-mmdiameter culture dishes and incubated overnight at 37° C. Subsequently,cells were pre-treated with 5 μM PPF for 24 hours and then eithertreated with 250 μM TMZ for 24 hours or exposed to 2Gy radiation doseusing a RS 2000 X-ray irradiator. Following combination therapy, cellswere trypsinized, counted, and plated in a 6-well culture dish atdensities of 100, 250, and 500 cells per well in triplicate. Cells wereincubated for 12 days then fixed, stained with 0.5% crystal violetsolution, and counted manually by blinded observers. The inventors'results demonstrate that treatment with PPF in combination with TMZsignificantly decreased the surviving fraction of T98G and GBM43 cellswhen compared to TMZ treatment alone (FIG. 26A). Similarly, combinationtreatment with PPF and 2Gy radiation significantly decreased thesurviving fraction of GBM cells when compared to 2Gy radiation alone(FIG. 26A). Next, to test whether the decrease in survival was due to anincrease in apoptosis, the inventors isolated lysate from T98G gliomacells after treatment with vehicle, PPF alone, TMZ alone, and PPF incombination with TMZ and immunoblotted for cleaved PARP. The inventorsfound that T98G cells exposed to the combination treatment of PPF andTMZ showed an increase in cleaved PARP as compare to TMZ treatment alone(FIG. 26B). Since TROY-mediated therapeutic resistance is dependent uponactivation of the AKT and NF-κB signaling pathways (See Reference 42),the inventors investigated the effect of PPF on TROY survival signaling.T98G and GBM43 cells were treated with PPF, lysed, and thenimmunoblotted to assess the activation of AKT and NF-κB. PPF effectivelydecreased AKT and NF-κB phosphorylation in both cell lines (FIG. 26C).These data validate that PPF inhibits TROY survival signaling pathwaysand augments the efficacy of TMZ in GBM.

Referring now to FIGS. 27A and 27B: The inventors next examined whetherPPF inhibited TROY dependent glioma cell invasion. T98G, GBM10, andGBM43 glioma cells were treated with PPF, and using a Matrigel invasionassay, the inventors demonstrated that treatment with 5 μM PPFsignificantly inhibited glioma cell invasion in vitro (FIG. 27A).Briefly, 5.0×10⁵ glioma cells were seeded in 100-mm diameter culturedishes and incubated overnight at 37° C. Subsequently, cells were serumstarved for 16 hours at 37° C. Cells were then harvested, re-suspendedin growth factor reduced Matrigel (Becton Dickinson) (1.0×10⁵ cells/50uL), added in triplicates to collagen-coated transwell chambers, andallowed to invade through Matrigel in presence of 10% FBS and/or 5 μMPPF. After incubation for 24 hours at 37° C., non-invaded cells werescrapped off the upper side of the membrane and cells invaded to theother side of the membrane were fixed with 4% paraformaldehyde (PFA)(Affymetrix) and stained with DAPI (Invitrogen). Nuclei of invaded cellswere counted in five high power fields (HPF) with a 20× objective.TROY-mediated invasion is induced, in part, through activation of Rac1,and the inventors' results showed that PPF inhibited Rac1 activation inT98G cells (FIG. 27B) (See Reference 43).

Referring now to FIGS. 28A and 28B: The inventors investigatedlamellipodia formation and membrane ruffling. Briefly, glioma cells wereplated onto 10-well glass slides pre-coated with 10 μg/ml laminin at thedensity of 3000 cells/well for 24 hours at 37° C. Subsequently, cellswere serum starved for 16 hours. The cells were pre-incubated with 5 μMPPF or vehicle for 1 hour prior to 10% FBS stimulation for 5 min. AfterFBS stimulation, cells were fixed in 4% PFA, permeabilized with 0.1%Triton X-100, and incubated with Alexa Fluor® 555 Phalloidin(Invitrogen) to stain for F-actin. Slides were mounted with ProLong®reagent with DAPI and imaged using a Zeiss LSM 510 microscope. For eachexperimental condition, at least 12 images were randomly taken.Lamellipodia were traced using ImageJ software. For each cell, thefraction of the cell perimeter that displayed lamellipodia wascalculated. The inventors evaluated lamellipodia formation after PPFtreatment in T98G, GBM10, and GBM43, cells, and showed that PPFsignificantly decreased lamellipodia formation and membrane ruffling(FIGS. 28A and 28B).

REFERENCES

So as to reduce the complexity and length of the Detailed Specification,Inventors herein expressly incorporate by reference to the extentapplicable, all of the following materials.

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1-20. (canceled)
 21. A pharmaceutical composition for the treatment ofcancer, the pharmaceutical composition comprising: a first activeingredient, wherein the first active ingredient is propentofylline or apharmaceutically acceptable salt thereof; and a second activeingredient, wherein the second active ingredient is a chemotherapeuticagent selected from the group consisting of: cis-diamminedichloroplatinum (II) (cisplatin), doxorubicin, 5-fluorouracil, taxol,topoisomerase inhibitor, temozolomide, and a targeted therapy drug. 22.The pharmaceutical composition of claim 21, wherein the topoisomeraseinhibitor is selected from the group consisting of: etoposide,teniposide, irinotecan, and topotecan.
 23. The pharmaceuticalcomposition of claim 21, wherein the targeted therapy drug is selectedfrom the group consisting of: an inhibitor of signaling through TROY(TNFRSF19) activation, bevacizumab, gefitinib, erlotinib, cetuximab,lapatinib, panitumumab, vandetanib, afatinib, icotinib, zalutumumab,nimotuzumab, and matuzumab.
 24. The pharmaceutical composition of claim23, wherein the inhibitor of signaling through TROY (TNFRSF19)activation is selected from the group consisting of: Pyk2 inhibitors,Rac1 inhibitors, Dock180 inhibitors, and Dock7 inhibitors.
 25. Thepharmaceutical composition of claim 21, wherein the second activeingredient is selected from the group consisting of: Pyk2 inhibitors,Rac1 inhibitors, Dock180 inhibitors, Dock7 inhibitors, temozolomide, andbevacizumab.
 26. The pharmaceutical composition of claim 21, wherein thesecond active ingredient is selected from the group consisting of: anantibody against TROY that inhibits TROY, Pyk2 shRNA, Rac1 siRNA,Dock180 siRNA, Dock7 siRNA, temozolomide, and bevacizumab.
 27. Thepharmaceutical composition of claim 21, wherein the second activeingredient is temozolomide.
 28. The pharmaceutical composition of claim21 further comprising at least one pharmaceutically acceptable carrier.29. The pharmaceutical composition of claim 21, wherein the compositiontreats glioblastoma.
 30. The pharmaceutical composition of claim 28,wherein the propentofylline or a pharmaceutically acceptable saltthereof is in in an amount sufficient to inhibit TROY expression, butthe amount is less than sufficient to substantially reduce viability ofglioblastoma cells.
 31. The pharmaceutical composition of claim 29,further comprising an anxiolytic agent.
 32. The pharmaceuticalcomposition of claim 21, further comprising an antiemetic ingredient.33. The pharmaceutical composition of claim 21, further comprising ahematopoietic colony stimulating factor.
 34. The pharmaceuticalcomposition of claim 21, further comprising an analgesic agent.